Variant-type carbohydrate hydrolase, variant gene of the enzyme and method for producing oligosaccharide using the enzyme

ABSTRACT

There is disclosed a variant-type carbohydrate hydrolase that has been increased transglycosylation activity by substituting another amino acid residue for the tyrosine residue that is present in the active center of the hydrolase, which hydrolase is an amylase or an enzyme analogous to amylase; a gene or a DNA sequence of the carbohydrate hydrolase with mutation introduced into the base sequence that encodes the tyrosine residue; and a vector or a transformant which comprises the DNA sequence. There is also disclosed a method for producing a variety of oligosaccharides and the like by using the variant-type carbohydrate hydrolase.

This application is a continuation of application Ser. No. 08/204,656,filed on Mar, 2, 1994, now U.S. Pat. No. 5,538,882 the entire contentsof which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a variant-type carbohydrate hydrolasethat has been increased in transglycosylation activity. In particularthis invention relates to an amylase or another carbohydrate hydrolaseanalogous to amylase, wherein the tyrosine residue is substituted withanother amino acid residue, which tyrosine residue constitutes theactive center of the enzyme. Furthermore, the present invention relatesto a gene or a DNA sequence with a mutation introduced into the basesequence that encodes the tyrosine residue of the carbohydratehydrolase; it relates to a variant enzyme in which the tyrosine residueof the carbohydrate hydrolase is substituted; it relates to a vector ora transformant which comprises the DNA sequence; and it relates to amethod for producing a variety of oligosaccharides and the like by usingthe variant enzyme.

BACKGROUND OF THE INVENTION

In recent years, as artificial sweeteners that can replace sugar withoutcausing tooth decay and as materials for functional foods, a variety ofoligosaccharides of various polymerization degrees have drawn attentionand have been put to practical use.

The present inventors have already reported that transglycosylationactivity is elevated in a variant-type α-amylase (SfamyW84L), in which aleucine residue is substituted for the tryptophan residue at position 84of the amino acid sequence of the α-amylase (Sfamy) which is obtainedfrom the yeast Saccharomycopsis fibuligera; and that a method forproducing a malto-oligosaccharide of a polymerization degree of 7 ormore, using the said variant-type α-amylase (unexamined Japanese PatentPublication No. 108386/1992). However, it has not yet been made clearthe mechanism by which the transglycosylation activity of the enzymeSfamy W84L is elevated. Thus, no method has yet been found for elevatingthe transglycosylation activity of Sfamy and other carbohydratehydrolases in a rational manner.

BRIEF SUMMARY OF THE INVENTION

The present inventors have made intensive investigations aimed atrationally increasing the transglycosylation activity of carbohydratehydrolases. As a result, the inventors have found that the tyrosineresidue corresponding to the Y83 residue of the Sfamy is completelypreserved original spatial arrangement at a homologous position innumerous carbohydrate hydrolases analogous to amylase, such as α-amylaseand cyclomaltodextrin glucanotransferase. Further, the inventors havefound that these tyrosine residues are present in the active centers ofthe enzymes, and that the phenolic OH group of the tyrosine residues maybe considered to have a definitely important function to provide H₂ Omolecules to a reaction intermediate during each enzyme's enzymaticreaction. Therefore, the inventors have found that by substitutinganother amino acid residue for the tyrosine residue, therebydeteriorating the function of the tyrosine residue during hydrolysis,the transglycosylation activity of the carbohydrate hydrotases can beincreased.

The present invention has been achieved based on the above findings.

Therefore, an object of the present invention is to provide avariant-type carbohydrate hydrolase that has been increased intransglycosylation activity, in particular to provide a hydrolasesubstituted another amino acid residue for the tyrosine residue that ispresent in the active center of the hydrolase, which hydrolase is anamylase or another carbohydrate hydrolase analogous to amylase.

Another object of the present invention is to provide a gene withmutation introduced into the base sequence that encodes the tyrosineresidue of the carbohydrate hydrolase.

Further, another object of the present invention is to provide a methodfor producing a variety of oligosaccharides and the like by using thevariant-type carbohydrate hydrolase that has been increased intransglycosylation activity.

Still another object of the present invention is to provide a method forrationally increasing the transglycosylation activity of enzyme that isan amylase or an enzyme analogous to amylase.

Other and further objects, features, and advantages of the inventionwill appear more evident from the following description, taken inconnection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a drawing that comparatively shows the amino acid sequences(SEQ ID NOS.:11-16) in three preserved-regions immediately next to theamino terminus of wild-type Sfamy and wild-type cyclomaltodextringlucanotransferase (CGTase).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is to provide:

1. A variant-type carbohydrate hydrolase, selected from a groupconsisting of α-amylase and cyclomaltodextrin glucanotransferase,wherein the hydrolase's tyrosine residue, which is present in the activecenter of the enzyme, and which functions to provide H₂ O molecules to areaction intermediate, is substituted (replaced) with another amino acidresidue;

2. The variant-type carbohydrate hydrolase according to the above item1, wherein the tyrosine residue is present within a 5 angsttoms rangefrom three acidic amino acid residues which are immediate catalyticresidues, and wherein the tyrosine residue has a function to provide H₂O molecules to a reaction intermediate;

3. A variant α-amylase, wherein the tyrosine residue at position 83 ofthe amino acid sequence of the α-amylase from Saccharomycopsisfibuligera is substituted with a phenylalanine, SEQ ID NO.:2, atryptophan, SEQ ID NO.:4, a leucine, SEQ ID NO.: 6, or an asparagineresidue, SEQ ID NO.: 8;

4. A variant α-amylase gene, wherein the base sequence at positions 247to 249 of said gene (positions 325 to 327 of the structural gene) in theα-amylase gene from Saccharomycopsis fibuligera is mutated into one ofTTC SEQ ID NO.:1, TTG SEQ ID NO.:3, CTC SEQ ID NO.:6, or AAC SEQ IDNO.:7;

5. A method for producing a malto-oligosaccharide of a polymerizationdegree of 7 or more, comprising subjecting a substratemalto-oligosaccharide to transglycosylation reaction with the variantα-amylase according to the above variant in item 3;

6. A variant cyclomaltodextrin glucanotransferase, wherein the tyrosineresidue at position 100 of the amino acid sequence of thecyclomaltodextrin glucanotransferase, SEQ ID NO.:10 from Bacillusmacerans is substituted with a tryptophan residue;

7. A variant cyclomaltodextrin glucanotransferase gene (SEQ ID NO.: 9),wherein the base sequence at positions 299 to 300 of said gene(positions 380 to 381 of the structural gene) of the cyclomaltodextringlucanotransferase from Bacillus macerans is mutated into GG; and

8. A method for producing oligosaccharides containing higherconcentrations of α-maltotripyranosyl-β-D-fructofuranoside andα-maltotripyranosyl-β-D-fructofuranoside, comprising subjectingsubstrates of starch and sucrose to transglycosylation reaction with thevariant cyclomaltodextrin glucanotransferase according to the abovevariant in item 6.

The present invention will now be explained in details hereinbelow.

The present inventors have found that substitution of the tryptophanresidue at position 84 of the variant-type SfamyW84L which alreadyreported, might have an influence on the adjacent tyrosine residue (Y83)at position 83, resulting in a modification of the Y83 residue'sfunction, to thereby increase the transglycosylation activity. In otherwords, it has been found that the principal factor for increasing thetransglycosylation activity is based on eliminating the function of theY83 residue.

Conventionally, it has been known as a general empirical rule that notonly the species but also the relative spatial geometry of an amino acidresidue, which residue is most important for expression of the activityof an enzyme, is kept as it is in analogous enzymes, despite that theycome from different organisms.

Examination was conducted to identify where the tyrosine residuecorresponding to the Y83 residue of Sfamy was positioned-on the stericstructure in the α-amylases enzymes such as Sfamy, derived from 10species of organisms. As a result, it was found that the configurationof the tyrosine residue is completely preserved in all of the 10 typesof α-amylases.

Ten types of α-amylases from the following organisms were examined:

1. Actinomyces (Stremtomyces hygroscopicus);

2. Pig pancreas;

3. Bacillus subtilis;

4. B. ciculans;

5. Saccharomycopsis fibuligera;

6. Aspergillus oryzae;

7. B. stearothermophilus;

8. B. lichenformis;

9. B. amyloliguefaciens; and

10. barley.

Based on the data concurrently demonstrating the primary sequences ofthe 10 types of α-amylases from the different organisms, comparativeexamination was done on the steric structure of the enzyme Sfamy, todetermine how the amino acid residues constituting the active site ofthe enzyme were conserved. Consequently, it was found that one Tyrresidue corresponding to the Y83 of Sfamy was present near the three,direct catalytic residues (two Asp residues and one Glu residue), i.e.within a 5 angstroms range from these residues, and that these fourresidues were completely preserved original spatial arrangement in the10 types of α-amylase proteins described above, to form each enzymaticactive center. Thereby, the tyrosine residue corresponding to the Y83residue of Sfamy may possibly have an important function through itsphenolic OH to provide H₂ O molecules to the intermediate of a enzymaticreaction.

Then, attention was focused on the Tyr residue (Y) forming the enzymaticactive center in the α-amylases. Examination was conducted to determineif the Y residue might be present in the following cyclomaltodextringlucanotransferase, which is analogous to α-amylase. Using Sfamy as anα-amylase, comparison was made of the amino acid sequences of the twoenzymes, i.e. Sfamy and the below-mentioned CGTase.

CGTase: cyclomaltodextrin glucanotransferase, derived from Bacillusmacerans.

From the results of the comparison of the primary amino acid sequencesof the two enzymes, six highly preserved homologous regions wereidentified. Among the six regions highly preserved spatial arrangement,three preserved regions immediately next to the amino terminus(N-terminus) have their amino acid sequences shown in FIG. 1.

The Y residue in focus, corresponding to the Y83 residue of the enzymeSfamy, is present in region 1, and the residue is also preserved at thehomologous position in cyclomaltodextrin glucanotransferase, as shown inthe confined part in region 1 cf FIG. 1, SEQ ID NO.:11and SEQ ID NO.:12.It was found that in cyclomaltodextrin glucanotransferase, the Tyrresidue was also present in the proximity of three directly catalyticresidues, i.e. two Asp residues and one Glu residue, namely within 5angsttoms from these residues, as in Sfamy as described above. Thereby,the tyrosine residue corresponding to the Y83 residue of Sfamy waspresent at position 100 in CGTase.

Furthermore, FIG. 1 shows clearly that these three highly preservedregions were arranged at nearly equal intervals.

Based on the findings described above and the fact that the amino acidresidues (Tyr residue, Glu residue, and two Asp residues) being presentin these three regions form an active center of the enzyme in α-amylase,it is considered that the completely preserved Y residue that is presentin region 1 and that corresponds to the Y83 residue of Sfamy, may bepresent in the active center of the enzyme in cyclomaltodextringlucanotransferase, to perform a definitely important function in thehydrolysis of substrates. Also, it is suggested that the tyrosineresidue functions through its phenolic OH group to provide H₂ Omolecules to a reaction intermediate during enzymatic reaction.

These findings suggest that the transglycosylation activity of the twotypes of enzymes analogous to amylase, i.e. Sfamy and CGTase, can beincreased by substituting the tyrosine residue that constitutes theactive center of each enzyme with another amino acid residue.

In the present invention, the "another amino acid residue" is introducedto lower the hydrolysis activity of the carbohydrate hydrolase. Theanother amino acid residue is an amino acid residue which has nophenolic OH group. Every amino acid other than tyrosine among 20 typescf natural amino acid has no phenolic OH group, and can be used as theanother amino acid residue.

A method for preparing a variant enzyme of the present invention, andthe effects on the enzyme activity of the substitution of the tyrosineresidue present in the active center of the variant enzyme are describedfirst.

In order to elucidate the function of the Y residue that is present inthe region 1 of FIG. 1, which Y residue is also present in theabove-mentioned two types of enzymes that are analogous to amylase,variant enzymes of the two enzymes (Sfamy and CGTase) were prepared,wherein the Y83 residue of Sfamy and the Y100 residue of CGTase weresubstituted with another amino acid residue, respectively. Then,examination was conducted to elucidate the effects of this substitutionon the carbohydrate hydrolysis activity and the transglycosylationactivity of these enzymes.

1. Variant-type Sfamy

As one example of α-amylase, variant-type enzymes were prepared, whereinthe Y83 residue of Sfamy was substituted with one of F, W, L, or Nresidue, to assess the transglycosylation activity.

The Kunkel method was employed for preparing variant genes.

From a plasmid pSfα1 that carried the gene of the enzyme Sfamy, anEcoRI-PstI fragment (2.5 kb) containing the Sfamy structural gene wassubcloned into the multi-cloning site of M13 phage. An M13 phagereplicated form (RF) DNA containing the objective DNA fragment wasinfected into a uracil-DNA glycosylase (ung) defective- E. coli strainCJ236, to recover the phage particles from the culture supernatant ofthe E. coli strain. From the recovered phage particles, uracilsingle-stranded DNA (minus (-) chain) was obtained. By annealing thesingle-stranded DNA with each of the synthetic oligonucleotidescontaining mutation in the coding region that encoded the Y residue, andthereafter by reacting T₄ DNA polymerase with the annealed product, torepair the single strand (plus (+) chain), each double-stranded DNA(U-RF DNA) was prepared. After introducing each of the DNAs into ung⁺ E.coli , the phage particle was recovered from the E. coli culturesupernatant, to prepare each single-stranded DNA. By analyzing the basesequence of the single-stranded DNAs, RF DNAs with the objectivemutation were selected.

A 2.5 kb EcoRI-PstI fragment cleaved out from each of the RF DNAs wasinserted into and ligated with the multi-cloning site of a yeastexpression vector YEp351, to prepare each of expression vectors of thevariant enzymes, i.e. pSA5Y83F, pSA5Y83W, pSA5Y83L, and pSA5Y83N.

Through the transformation of each of these expression vectors in strainKK4 of the baker's yeast Saccharomyces cerevisiae, transformantsKK4:pSA5Y83F, KK4:pSA5Y83W, KK4:pSA5Y83L, and KK4:pSA5Y83N wereobtained. Similarly, a transformant KK4:pSA5 carrying the gene of thewild-type enzyme was also prepared. After culturing these transformantsin the YPD medium, various purification procedures were employed tosupernatant of the culture, to prepare purified variant enzymes, i.e.SfamyY83F, SfamyY83W, SfamyY83L, and SfamyY83N, as well as the purifiedwild-type enzyme, Sfamy.

The four variant enzymes: SfamyY83F, SfamyY83W, SfamyY83L, andSfamyY83N, prepared in accordance with the present invention, allexhibited far stronger transglycosylation activity than carbohydratehydrolysis activity, to individually prepare various characteristicproducts depending on the polymerization degree of each startingsubstrate.

When maltoheptaose (G₇) was a substrate, for example, it was partiallydecomposed into lower molecules, but was mostly transformed into G₁₀,G₁₁ and G₁₂. When maltopentaose (G₅) was a substrate, transglycosylationproducts, G₇ and G₈, were produced; from maltohexaose, (G₆), G₉ wasproduced.

Through separation and purification procedures, such as gel filtration,malto-oligosaccharides of longer chains by specific chain lengths thanthose of these substrates were produced from the resulting reactionmixtures.

The optimum conditions for the above transglycosylation enzyme reactionsare approximately a temperature of 30° C. and pH 5.5, for example. Thereaction time varies depending on the polymerization degree andconcentration of a substrate and the enzyme concentration, but thegeneral satisfactory reaction time is 15 minutes.

The physicochemical properties of the variant-type α-amylases will nowbe described hereinbelow.

(1) Function

The variant enzymes of the present invention exhibit far strongertransglycosylation activity than the wild-type enzyme, so the variantenzymes exhibit strong transglycosylation activity at a lower substratelevel (2 mM), at which level the wild-type enzyme does not exhibittransglycosylation activity. When the variant enzymes react withmalto-oligosaccharide, malto-oligosaccharides of a longer chain thanthat of the initial substrate are prepared. Various characteristicproducts are prepared, depending on the polymerization degree of eachstarting substrate. When maltoheptaose (G₇) is a substrate, for example,it is partially decomposed into lower molecules, but is mostlytransformed into G₁₀, G₁₁, and G₁₂. When maltopentaose (G₅) is asubstrate, G₇ and G₈ are prepared; from maltohexaose (G₆) as asubstrate, G₉ is prepared.

(2) Substrate specificity

The variant enzymes of the present invention react well with starch,glycogen, and malto-oligosaccharide, but they do not react withpullulan, dextran, or cellulose.

(3) Optimum pH and stable pH range

The optimum pH for the variant enzymes is from pH 5.4 to 5.6 at 40° C.,and the stable pH range is from pH 4 to 10.

(4) Optimum temperature

The optimum temperature for the variant enzymes is at 50° C. at pH 5.5.

(5) Inactivating conditions

Under heating at 70° C. and pH 5.5 for 10 minutes, the remainingactivity of each of the variant enzymes is at a ratio of 10%. Theenzymes are unstable at pH 4 or less, or at pH 10 or more.

(6) Inhibition, activation, and stabilization

The variant enzymes are inhibited in the presence of mercury, lead, andEDTA. The enzymes are stabilized by calcium ion.

(7) Assay of transglycosylation activity

Terminating the enzyme reaction after the prescribed period of time, thereaction products were spread out and separated by paper chromatography;then the products were sensitized with glucoamylase treatment, followedby development with silver nitrate. Furthermore, the molecular speciesof individual spots were identified and analyzed quantitatively with adensitometer; the change of the products through the enzyme reaction wasanalyzed over time, to determine transglycosylation activity.

(8) Molecular weight of the enzymes

The molecular weight of each of the variant enzymes is 51, 000 (by diskgel electrophoresis).

2. Variant-type CGTase

A variant enzyme (CGTaseY100W) was prepared, wherein the Y100 residue(corresponding to Y83 residue of Sfamy) of cyclomaltodextringlucanotransferase (CGTase) derived from Bacillus macerans, which is anenzyme analogous to α-amylase, was substituted with a W residue. Thetransglycosylation activity of CGTaseY100W was determined.

First, CGTase gene was cloned.

The chromosomal DNA from B. macerans IAM 1243 strain was treated withSau3AI and Hind III; then it was ligated with pBR322; then it was theninserted into E. coli HB101.

The cloning strain carrying CGTase gene was detected by iodine starchreaction on an agar medium containing starch. Consequently, arecombinant plasmid pMAC1 with a 4 kb DNA containing CGTase geneinserted was prepared. The pMAC1 was partially digested with Sau3AI;then it was inserted and ligated in the BamHI site of a plasmidpHY300PLK, to prepare pMAA1. The plasmid was introduced in anα-amylase-defective B. subtilis strain 207-25, to prepare a transformantstrain 207-25:pMAA1; then it was tetracycline-resistant and had haloformation potency during iodine starch reaction on starch-agar medium.Purified CGTases obtained from the culture supernatants of B. maceransIAM 1243 strain and B. subtilis 207-25:pMAA1 were subjected toenzyme-reaction with soluble starch, and the resulting enzyme-reactionproducts were analyzed by paper chromatography. The main reactionproducts of the two enzyme reactions were α-cyclodextrin, and the tworeactions could not be discriminated from each other in terms of theirenzymatic natures. Furthermore, the base sequence of the DNA fragmentinserted into the plasmid pMAA1 was determined. The base sequence of thestructural gene of CGTase, which base sequence consisted cf 2142 bp, wasdetermined, and translated into the amino acid sequence of CGTase.

Variant gene was prepared by the Kunkel method.

First, an XbaI-EcoRI fragment (2.5 kb) containing the CGTase structuralgene was subcloned from pMAA1 into M13 phase, and the M13 phage RF DNAcontaining the objective DNA fragment was infected into a ung⁻ E. colistrain CJ236. The phage particle was then recovered from the culturesupernatant of the E. coli strain, to prepare uracil single-stranded DNA(minus (-) chain). By annealing the single-stranded DNA and a syntheticoligonucleotide having a mutation in the coding region encoding the Y100residue, and then repairing the single strand (plus (+) chain), adouble-stranded DNA (U-RF DNA) was prepared. After inserting the DNAinto ung⁺ E. coli , the phage particle was recovered from the E. coliculture supernatant, to prepare RF DNA.

After analyzing the base sequence of the RF DNA, a DNA fragment(XbaI-EcoRI fragment of 2.5 kb) carrying the objective mutation wasincised out; then it was inserted and ligated in the XbaI-EcoRI site ofthe B. subtilis expression vector, PHY300PLK, whereby a variant enzymeexpression vector pMAA1Y100W was prepared.

Through the transformation of these expression vectors, whichindividually carried the wild-type enzyme gene and the variant-typeenzyme gene, into the B. subtilis 207-25 strain, transformants B.subtilis 207-25:pMAA1 and 207-25:pMAA1 Y100W were obtained.

After culturing these transformants in the NB medium supplemented with1% soluble starch, various purification procedures were employed torecover the purified wild-type enzyme and the purified variant-typeenzyme, i.e. CGTase Y100W, from the culture supernatants.

Besides cyclodextrin production via CGTase action with starch, CGTasetransfers malto-oligosaccharide into sugar receptors if the receptormolecules are present along with starch. The variant enzyme,CGTaseY100W, prepared in accordance with the present invention,exhibited far stronger transglycosylation activity than the wild-typeenzyme.

When CGTase Y100W reacts with a solution containing 5% soluble starchand 5% sucrose, for example, the production of a small amount ofcyclodextrin is observed at the initial stage, but the preparedcyclodextrin is decomposed again and is eliminated at the later stage.In this reaction, 50% of the existing sucrose is utilized as thereceptor for the action of transglycosylation. In the oligosaccharidesprepared, G₂ F (α-maltopyranosyl-β-D-fructofuranoside) and G₃ F(α-maltotripyranosyl-β-D-fructofuranoside) are contained at ratios of21% and 12%, respectively, in addition to sucrose at a ratio of 24%.Thus, the use of the variant enzyme CGTase Y100W enables highlyefficient production of an oligosaccharide containing G₂ F and G₃ F.

The optimum conditions for reacting the transglycosylase enzyme areapproximately 50° C. and pH 5.5, for example. The reaction time variesdepending on the concentration of a substrate and the enzymeconcentration. Generally, a satisfactory reaction time is 15 minutes.

The physico-chemical properties of the variant-type CGTase will now bedescribed hereinbelow.

(1) Function

The variant enzyme of the present invention exhibits far strongertransglycosylation activity than the wild-type enzyme. When the variantenzyme reacts with soluble starch in the presence of sucrose as thereceptor, a glucosyl group, a maltosyl group, and a maltotriosyl groupare transferred into the receptor (sucrose), to preparemalto-oligosaccharide derivatives, which are of various chain lengthsand which contain fructose at the reducing end thermini; this derivativeproduction is more efficient than the same reaction involving thewild-type enzyme.

(2) Substrate specificity

The variant enzyme reacts well with starch and malto-oligosaccharide,but it does not react with pullulan, dextran, or cellulose.

(3) Optimum pH and stable pH range

The optimum pH of the variant enzyme is from pH 5.4 to 5.6 at 40° C.,and the stable pH range is from pH 3 to 1.

(4) Optimum temperature

The optimum temperature of the variant enzyme is 60° C. at pH 5.5.

(5) inactivating conditions

Under heating at 70° C. and pH 5.5 for 10 minutes, the remainingactivity of the variant enzyme is at a ratio of 30%. The enzyme isunstable at pH 3 or less, or at pH 11 or more.

(6) Inhibition, activation, and stabilization

The variant enzyme is inhibited in the presence of mercury, lead, andEDTA. The enzyme is stabilized by using calcium ion.

(7) Assay of transglycosylation activity

Terminating the enzyme reaction after the prescribed period of time, thereaction products were spread out and separated by paper chromatography;then the products were sensitized with glucoamylase treatment, followedby development with silver nitrate. Furthermore, the molecular speciesof individual spots were identified and analyzed quantitatively with adensitometer; the change of the products through the enzyme reaction wasanalyzed over time, to determine transglycosylation activity.

(8) Molecular weight of the enzyme

The molecular weight of the variant enzyme is 74,000 (by disk gelelectrophoresis).

Each of the variant-type carbohydrate hydrolases in accordance with thepresent invention exhibits far greater carbohydrate hydrolysis activitythan the wild type, due to the mutation of the tyrosine residue presentin the active center of the enzyme. The use of each of the variant-typeenzymes of the present invention enables the production of a specificmalto-oligosaccharide of a high polymerization degree of 7 or more, orthe production of an oligosaccharide containing high concentrations ofG₂ F and G₃ F, both at a high yield. Hence, the variant enzymes canpreferably be used for large-scale industrial production of a variety ofoligosaccharides.

The variant genes of the present invention enable the preparation ofvariant enzymes, belonging to the group of amylase or its analogouscarbohydrate hydrolase, that are rationally provided withtransglycosylation activity essential for the production of usefulenzymatic products, such as malto-oligosaccharide.

Next the present invention will be described in detail in accordancewith examples, but the invention is not limited to these examples.

EXAMPLE 1

A method for preparing variant-type Sfamys

Variant genes were prepared, wherein the structural gene of Sfamy wasmutated by site-directed mutagenesis.

The variant genes were prepared according to the Kunkel procedure.

From a plasmid pSfα1 carrying the Sfamy gene, a 2.5 kb EcoRi-PstI DNAfragment carrying the Sfamy structural gene was inserted in and linkedwith the multi-cloning site of M13 phage mp18. The M13 phage replicatedform (RF) DNA containing the objective DNA fragment was infected in auracil-DNA glycosylase (ung)-defective E. coli strain CJ236. Theinfected bacterium was cultured in the LB medium (1% Bactotripton, 0.5%yeast extract, 0.5% NaCl, and 0.1% glucose). From the culturesupernatant, the phage particle was recovered and extracted with phenol,to yield uracil-containing single-stranded DNA (minus (-) chain).

In order to substitute the Y83 residue (codon; TAC) with respectivelyeach of phenylalanine (codon; TTC), tryptophan (codon; TGG), leucine(codon; CTC), and asparagine (codon; AAC), four types of 21-meroligonucleotides were synthesized, in which the underlined codon in theoligonucleotide sequence 5' TAT CAT GGT TAC TGG ATG AAG 3' ,SEQ IDNO.:17, was substituted with each of the codons for the above-mentionedobjective four amino acids. By annealing the single-stranded DNAcontaining uracil with each of the synthetic oligonucleotides, followedby effecting the action of T₄ DNA polymerase and T₄ DNA ligase, eachsingle strand (plus (+) chain) was repaired, to prepare eachdouble-stranded DNA (U-RF DNA). By inserting each of the U-RF DNAs intoung⁺ E. coli strain (MV1190 strain), each DNA strand containing uracilwas decomposed with the ung to recover the phage particle in replicationor RF DNA from the DNA strand that had a mutation introduced.Specifically, each U-RF DNA was introduced into MV1190 strain with DNAreceptivity enhanced by the calcium chloride method; the U-RF DNA wasthen inoculated on the LB solid medium together with the indicatorbacterium (MV1190 strain), followed by overnight incubation at 37° C.The phage particle from the plaques that had grown by the next day wastransferred into the TE solution (10 mM Tris-HCl buffer, pH 8.0,containing 1 mM EDTA) and was kept at 4° C. Simultaneously, four clonesamong them were separately infected in the MV1190 strain in 2 ml of LBliquid medium, followed by culturing for 7 hours at 37° C. The culturemedium was centrifuged, and from the resulting supernatant, the phageparticle was recovered as the precipitate in polyethylene glycol; theparticle was then treated with phenol, to isolate each single-strandedDNA. The base sequences of the single-stranded DNAs were thendetermined, to selectively isolate phage clones that had an accuratelyinserted mutation.

The base sequences of the variant genes were determined by the dideoxymethod. Ten types of 17-mer oligonucleotides (P₁ to P₁₀) weresynthesized, which have complementarity with the minus chain of thestructural gene, and which cover the entire structural gene. Among them,an oligonucleotide P₂ (2.5 ng), which was located at the 5' terminus ofthe mutated site (position 325 to position 327 of the base sequence ofthe structural gene, i.e., positions 247 to 249 in SEQ ID NOS.:1, 3, 5,and 7), and which corresponded to position 267 to position 283i.e.,positions 189 to 205 in SEQ ID NOS.:1, 3, 5, and 7, was annealed with 1μg of each of the previously isolated single-stranded DNAs (minus (-)chains) under heating, with subsequent annealing. Using 5 μCi of [α-³²P]dCTP and a commercially available sequencing kit, elongation,labeling, and termination reactions of the plus chains of the resultingannealed products were carried out at 37° C.

Each of the samples was heated at 100° C. for 2 minutes, followed byrapid cooling in ice; then it was added to 8% acrylamide gel (thickness;0.2 mm, length; 50 cm) containing 8.3M urea, followed by electrophoresisin the TBE solution (45 mM Tris-borate buffer, pH 8.3 containing 1 mMEDTA) for 2 hours. After electrophoresis, each of the gels wastransferred and dried on a filter. Then, each of the filters was used toexpose an X-ray film at -80° C. for 3 hours to obtain an autoradiogram.By identifying the bands developed thereon sequentially, the basesequences were determined. Through the above procedure, phage cloneswere prepared, wherein a part of the base sequence, corresponding to thetyrosine (Y) codon at position 325 to position 327of the structuralgene, was respectively substituted with phenylalanine (F), tryptophan(W), leucine (L), and asparagine (N) codons. As regards these four phageclones, by using the other nine types of synthetic oligonucleotides, thebase sequences of all of the structural genes were determined. It wasdemonstrated that no mutation was present except the objective mutation.These phage clones were designated as M13SfamyY83F, M13SfamyY83W,M13SfamyY83L, and M13SfamyY83N, respectively.

The base sequences, together with the amino acid sequences, are shown inSEQUENCE ID NOs.: 1, 3, 5 to 7, concerning the structural genes of thefour types of the variant-type Sfamy enzymes. In the above basesequences, base sequences encoding for an amino acid residue, thatreplaced the Y83 residue, are shown.

Expression vectors for yeast were prepared as follows. First, E. coliMV1190 strain was separately infected with the above-mentioned fourtypes of phage clones (M13SfamyY83F, M13SfamyY83W, M13SfamyY83L, andM13SfamyY83N). From the bacteria, each respective RF DNA was thenisolated by alkaline bacteriolysis; then it was subsequently treatedwith restriction enzymes EcoRI and PstI, and it was subjected to agaroseelectrophoresis, to yield a 2.5 kb DNA fragment carrying each of therespective variant α-amylase genes. Because each of the fragmentsoriginally contained a promoter region and a terminator region essentialfor expression of α-amylase, each of the fragments was inserted in andligated with the multi-cloning site of a yeast-E. coli shuttle vector,YEp351, which had been preliminarily digested with the same two types ofrestriction enzymes. The recombinant plasmids were used for transformingE. coli XL1-Bleu strain, and from the resulting bacteria of thetransformants, the plasmid DNAs were isolated by alkaline bacteriolysis.A part of each plasmid DNA was used to confirm that the objective 2.5 kbDNA fragment was inserted in the multi-cloning site. The plasmids wereindividually designated as pSA5Y83F, pSA5Y83W, pSA5Y83L, and pSA5Y83N.

Following the method of Hinnen et al., a baker's yeast (Saccharomycescerevisiae) strain KK4 was transformed with 3 μg of each of theserecombinant yeast expression vectors, to isolate four types oftransformants (KK4:pSASYS3F, KK4:pSA5Y83W, KK4:pSA5Y83L, andKK4:pSA5Y83N), using as the selective marker the leucine biosynthesisgene in the vectors. Wherein, the host yeast Saccharomyces cerevisiaestrain KK4 was a known yeast, with a gene marker attached (α, ura 3, his1/3, trp 1, leu 2, gal 80). Furthermore, the α-amylase gene of yeastSaccharomycopsis was inserted in the multi-cloning site of a yeastexpression vector YEp351 (see Yeast, 1986, 2, 163-169), to prepare theyeast expression vector pSA5.

Transformation of the baker's yeast (by the method of Hinnen et al.)will now be explained hereinafter. The KK4 strain was cultured overnightin the YPD liquid medium at 30° C., and 3 ml of the resulting yeast insolution was inoculated and cultured in 100 ml of the YPO medium at 30°C. for several hours. When the yeast reached a logarithmic growth stage(at a Klett figure of 180 to 200), the yeast were harvested bycentrifugation and washed in TE solution (40 ml), and the resultingyeast were suspended in the TE solution (6 ml). 2 ml of the suspensionwas transferred into an L-type test tube, followed by addition of 0.2Mlithium acetate (2 ml), with subsequent shaking at 30° C. for 1 hour.Then, glycerol was added (0.7 ml), after which 90 μg of each one of therecombinant plasmid DNA was added, with stirring; then the resultingmixture was kept at 30° C. for 30 minutes. Next, 4.5 ml of 70%polyethylene glycol #4000 was added to and mixed with the mixture, andit was kept at 30° C. for 1 hour. Then, the mixture was heated at 42° C.for 5 minutes, followed by rapid cooling at room temperature. Then, 10.5ml of water was mixed into the mixture. The yeast were harvested fromthe mixture with a centrifuge, washed again with 10.5 ml of water, andthen were collected. Finally, the yeast were suspended in 7.5 ml ofwater. 0.5 ml of the suspension, containing each one of the transformedcells respectively, was mixed in 2.5 ml of 0.7% soft agar, andinoculated and cultured in a solid synthetic selective medium,containing glucose as a single carbon source but without containingleucine, at 30° C. for several days, to isolate each of the transformedbaker's yeasts. As accession numbers FERM BP-134945471, FERM BP-5470,FERM BP- 5469, and FERM BP-5468, these transformants; i.e. KK4:pSA5Y83F,KK4:pSA5Y83W, KK4:pSA5Y83L, and KK4:pSA5Y83N, were deposited in theNational Institute of Bioscience and Human-Technology of the Agency ofIndustrial Science and Technology, at 1-3, Higashi 1-chome, Tukuba-shi,Ibaraki-ken, 305, Japan. These four transformants were deposited on Mar.2, 1993, and will have deposited to the institute for the period similarto those under the term of the Budapest treaty. All restrictions on theavailability to the public of the deposited materials will beirrevocably removed upon the granting of a patent. A similar expressionvector for the wild-type α-amylase (Sfamy) gene was prepared anddesignated as pSA5. The resulting transformant of pSA5 was designated asKK4:pSA5.

The transformant KK4:pSA5Y83F, containing the vector (pSA5Y83F) carryingthe gene to replace the Y83 residue of α-amylase with F, is a variantstrain of the above-mentioned KK4 strain. Similarly, KK4:pSA5Y83Wcarries the vector (pSA5Y83W) containing the gene to replace the Y83residue with W; KK4:pSA5Y83L carries the vector (pSA5Y83L) containingthe gene to replace the Y83 residue with L; and KK4:pSA5Y83N carries thevector (pSA5Y83N) containing the gene to replace the Y83 residue with N.

The expression and secretion of the wild-type and variant-typeα-amylases will next be explained hereinbelow, along with thepurification of these enzymes. The five types of transformed baker'syeasts carrying the wild-type gene or each variant-type Sfamy gene wereinoculated on the YPD liquid mediuJn (1 liter), and were shaken andcultured at 30° C. for 5 days. Subsequently, each culture supernatantwas obtained by centrifugation, and then to each was added 20 g of anionexchange resin, i.e. DE52-cellulose (manufactured by Wattmann, Co.),followed by agitation at 4° C. for 1 hour. Then the resin wasprecipitated and the supernatant was discarded, the remaining resin waswashed three times with 500 ml of 50 mM acetate buffer, pH 5.5,containing 2.5 mM calcium chloride. Then the resin was packed in a glasscolumn, followed by elution of proteins by a linear gradient of 1M NaCl.The enzyme activity of each fraction was assayed by iodine starchreaction. To a 16-ml portion of each active fraction was added ammoniumsulfate, to a final saturation degree of 80%; this was then centrifuged,to recover the precipitate. The precipitate dissolved in 1 ml of 50 mMacetate buffer (pH 5.5) was then applied to a hydrophobic chromatographycolumn, Butyl Toyopearl 650 (manufactured by TOSO, Co.), which hadpreviously been equilibrated with 50 mM acetate buffer saturated with40% ammonium sulfate, followed by elution and fractionation by a lineargradient of 40% to 0% ammonium sulfate. 10 ml of each of the activefractions was concentrated by ultrafiltration, by means of a membrane,YM30 (manufactured by Amicon, Co.), to a final volume of 0.2 ml; then itwas applied to a Superose 12 column (φ10×800 mm; manufactured byPharmacia, Co.) equilibrated with 50 mM acetate buffer for gelfiltration, to yield a wild-type enzyme sample or each variant-typeenzyme sample. By SDS-polyacryl amide gel electrophoresis, each of theseenzyme samples was at a respective single protein band; the top of thewild-type enzyme was at the same position as the top of the variant-typeenzymes. Further, it was identified by Western immunoblotting with amouse ascites antibody and ¹²⁵ I-protein A that the antigenicity of thewild-type enzyme agreed well with that of the variant-type enzymes. Bythe procedures described above, thus, the wild-type and variant-typeα-amylases were thoroughly purified.

It was identified that the obtained variant-type enzymes had thephysicochemical properties described above.

EXAMPLE 2

A method for preparing variant-type CGTase

CGTase gene was first cloned. The chromosomal DNA from B. macerans IAM1243 strain was treated with Sau3AI and Hind III, and linked withpBR322, which was then inserted into E. coli HB101.

The cloned strain carrying the CGTase gene was detected by iodine starchreaction on a starch-containing agar medium. As a result, a recombinantplasmid pMAC1 with a 4 kb DNA carrying the CGTase gene inserted wasprepared. The pMAC1 was partially digested with Sau3AI; then it wasinserted in and ligated with the BamHI site of the plasmid pHY300PLK, toprepare pMAA1. The plasmid was introduced in an α-amylase-defective B.subtilis strain 207-25, to prepare a transformant strain 207-25:pMAA1,which was tetracycline-resistant and had a halo formation potency duringiodine starch reaction on starch-agar medium. Purified CGTases obtainedfrom the culture supernatants of the B. macerans iAM 1243 strain and B.subtilis 207-25:pMAA1 were reacted with soluble starch. The resultingenzyme-reaction products were analyzed by paper chromatography. The mainreaction product from the two enzyme reactions was α-cyclodextrin, andthe two reactions could not be discriminated from each other withrespect to their enzymatic natures. Furthermore, the base sequence ofthe DNA fragment inserted into the plasmid pMAA1 was determined. Thebase sequence of the structural gene of CGTase, which gene consisted of2142 bp, was determined.

The resulting structural gene of CGTase was mutated by site-directedmutagenesis, to prepare a variant gene.

The variant gene was prepared by the Kunkel method.

From the plasmid pMAA1 carrying the CGTase gene, an XbaI-EcoRI fragment(2.5 kb) carrying the CGTase structural gene was inserted in and ligatedwith the multi-cloning site of M13 phage mp18. The M13 phage RF DNAcontaining the objective DNA fragment was infected in a ung⁻ E. colistrain CJ236. The infected bacterium was cultured in the LB medium. Fromthe culture supernatant, the phage particle was recovered and extractedwith phenol, to obtain uracil-containing single-stranded DNA (minus (-)chain).

In order to substitute the Y100 residue (codon; TAC) with tryptophan(codon; TGG), a 21-mer oligonucleotide was synthesized, in which theunderlined TAC codon in the oligonucleotide sequence 5' TAT CAC GGT TACTGG GCG AGG 3' , SEQ ID NO.:8 was substituted with a codon TGG. Byannealing the single-stranded DNA containing uracil with the syntheticoligonucleotide, followed by effecting the action of T₄ DNA polymeraseand T₄ DNA ligase, the single strand (plus (+) chain) was repaired toprepare a double-stranded DNA (U-RF DNA). By inserting the U-RF DNA intoung⁺ E. coli strain (MV1190 strain), the DNA strand containing uracilwas decomposed with the ung, to obtain the phage particle in replicationor RF DNA from the DNA strand with the mutation introduced.Specifically, the MV 1190 strain was transformed with U-RF DNA by thecalcium chloride method; then it was inoculated on the LB solid mediumtogether with the indicator bacterium (MV1190 strain), followed byincubation overnight at 37° C. The phage particle from the developedplaques was transferred into the TE solution and was kept at 4° C.Simultaneously, four clones among them were separately infected into theMV1190 strain in respective 2-ml portions of LB liquid medium, followedby culturing at 37° C. for 7 hours. From the resulting culture liquid,each respective phage particle was recovered as the precipitate inpolyethylene glycol; then it was treated with phenol, to separate thesingle-stranded DNA. The base sequence of this DNA was then determined,to select phage clones that had an accurately inserted mutation of TACcodon to TGG codon.

The base sequence of the variant gene was determined by the dideoxymethod. 14 Types of 17-mer oligonucleotides (Q₁ to Q₁₄) weresynthesized, which have complementarity with the minus (-) chain of thestructural gene, and which cover the entire structural gene. Among them,an oligonucleotide Q₃, which was located at the 5' terminus of themutated site (position 380 to Position 381 of the base sequence of thestructural gene i.e., positions 299 to 300 in SEQ ID NO.:9), and whichcorresponded to position 301 to position 317i.e., positions 220 to 236in SEQ ID NO.:9, was annealed with the previously isolatedsingle-stranded DNA (minus (-) chain) under heating, with subsequentannealing. Using [α-³² P]dCTP and a commercially available sequencingkit, elongation, labeling, and termination reactions of the plus chainof the resulting annealed product were done at 37° C. The sample washeated at 100° C. for 2 minutes, following by rapid cooling in ice; thenit was added to 8% acrylamide gel containing 8.3M urea, followed byelectrophoresis in the TBE solution for 2 hours. After electrophoresis,the gel was transferred and dried on a filter, to prepare anautoradiogram to determine the base sequence. Through the aboveprocedure, phage clones were prepared, wherein a part of the basesequence, corresponding to the tyrosine (Y) codon (TAC) at position 379to position 381of the structural gene, was substituted with a tryptophancodon (TGG). By using the other 13 types of synthetic oligonucleotides,the base sequences of all of the structural genes on the clones wereidentified. It was demonstrated that no mutation was present except theobjective mutation. The phage clone was designated as M13CGTaseY100W.

The base sequence, is shown in SEQUENCE ID NO: 9, concerning thestructural gene of the variant-type enzyme CGTaseY100W. In the abovebase sequence, the base sequence encoding the W residue, i.e., TGG,replaces the base sequence encoding the Y100 residue, i.e., TAC.

Next, E. coli MV1190 was infected with the M13CGTaseY100W. From theinfected bacteria, RF DNA was isolated by alkaline bacteriolysis; it wasthen treated with restriction enzymes XbaI and EcoRI, followed byagarose electrophoresis, to yield a 2.5 kb DNA fragment carrying thevariant CGTase gene. Because the fragment originally contained apromoter region and a terminator region essential for CGTase expression,the fragment was inserted in and ligated with the PLK site of B.subtilis vector, pHY300PLK, which had been preliminarily digested withthe same two types of restriction enzymes, to prepare pMAA1Y100W.

By transforming an α-amylase-defective B. subtilis strain 207-25 withthe plasmid pMAA1Y100W and the plasmid pMAA1 carrying the wild-typeCGTase gene, transformants 207-25:pMAA1 and 207-25:pMAA1Y100W wereprepared. The host bacterium B. subtilis 207-25 strain was a knownbacterium, with a gene marker attached (m₁₆₈ ⁻, hsrM, recE4, amyE07,aroI906, leuA8, and lys-21). The CGTase gene from B. macerance wasinserted into the multi-cloning site of a Bacillus vector pHY300PLK(Gene, 1984, 32, 129-135), to prepare a Bacillus vector pMAA1.

The bacterium Bacillus was transformed as follows B. subtilis strain207-25 was inoculated on a plate medium of Tryptose Blood agar base(TABA; manufactured by Difco, Co.) supplemented with glucose with 0.5%,followed by culturing overnight at 30° C. Young bacteria were scrapedand inoculated on 10 ml of the CI medium (1.4% K₂ HPO₄, 0.6% KH₂ PO₄,0.1% sodium citrate•2H₂ O, 5 mM MgSO₄ •7H₂ O, 0.5% glucose, 0.02%Casamino acid (manufactured by Difco), 50 μg/ml L-tryptophan, 50 μg/mlessential amino acid, 100 μg/ml essential base), to a final absorbance(at 660 nm) of about 0.1, following by shaking and culturing at 36° C.When the bacteria reached a logarithmic growth stage, they werecollected by centrifugation and were suspended in a two-fold volume ofthe CII medium (in which the contents of L-tryptophan, essential aminoacid, and essential base were one-tenth the same contents in the CImedium, but other ingredients were identical with the CI medium),followed by shaking and culturing for another 40 minutes; thenrecombinant plasmid DNA was added (10 μg), and culturing was continuedfor another 40 minutes. Then the bacteria were harvested bycentrifugation and were suspended in 1 ml of the LB medium. Thesuspension was inoculated on the TBAB medium supplemented withtetracycline (20 μg/ml) and 1% soluble starch, followed by overnightwarming at 36° C., to prepare a tetracycline-resistant transformant(207-25:pMAA1 Y100W).

The transformant (207-25:pMAA1 Y100W), containing a vector (pMAA1 Y100W)carrying the gene substituting the Y100 residue of CGTase with W, stillbelonged to the 207-25 strain.

The expression and secretion of the wild-type and variant CGTases willnext be explained hereinbelow, along with the purification of theenzymes. The two types of the transformants (207-25:pMAA1 and207-25:pMAA1Y100W) carrying the wild-type CGTase gene and thevariant-type CGTase gene were separately inoculated in 1 liter of the NBmedium (0.8% Nutrient Broth, manufactured by Difco, Co., 0.5% glucose, 5mM Ca(NO₃)₂) supplemented with 1% soluble starch, followed by shakingand culturing at 36° C. for 5 days. Subsequently, each of the culturesupernatants was obtained by centrufugation, and then to each was addedammonium sulfate, to a final saturation degree of 25%, and the resultingmixture was adjusted to pH 6.5 with an aqueous sodium hydroxidesolution. The solution was centrifuged and precipitates were discarded.Then, in the presence of 15% ammonium sulfate, pH 7.8, the resultingsolution was heated at 70° C. for 30 minutes. Next, to the solution wasadded rapidly cooled corn starch suspension, followed by agitationovernight at 4° C., to adsorb the CGTase onto the corn starch particles.Then, the CGTase-starch particle complex was recovered by centrifugationat 4° C. The CGTase was released from the complex, by keeping thecomplex at 40° C. for 1 hour in 50 mM acetate buffer, pH 5.5, containing2.5 mM calcium chloride. The released CGTase was dialyzed against 50 mMacetate buffer, pH 5.5, containing 25 mM calcium chloride. The solutionwas applied to a DE-52 cellulose column equilibrated with the samebuffer, followed by elution by a linear sodium gradient (0-1M) in thesame buffer. The enzyme activity of each fraction was assayed by iodinestarch reaction. A 10 ml portion of each active fraction wasconcentrated to a final volume of 0.2 ml by ultrafiltration, by means ofa membrane YM30 (manufactured by Amicon, Co.). Then it was applied to aSuperose 12 column (φ10×800 mm; manufactured by Pharmacia, Co.) whichhad been equilibrated with 50 mM acetate buffer, and gel filtration wascarried out to it, to yield wild-type CGTase and variant-type CGTase. BySDS-polyacrylamide gel electrophoresis, each of these enzyme samples wasat a respective unique protein band; the top of the wild-type enzyme wasat the same position as the top of the variant-type enzymes. Further, itwas identified that the antigenicity of the wild-type enzyme agreed wellwith that of the variant-type enzyme in a similar manner in Example 1.

It was also identified that the obtained variant-type enzyme had thephysicochemical properties described above.

EXAMPLE 3

Effects of replacing Sfamy Y83 residue on hydrolysis activity andtransglycosylation activity

In 400 μl of 50 mM acetate buffer, pH 5.5, containing 2.5 mM calciumchloride, a substrate maltoheptaose (G₇, 2×10⁻³ M) was reacted with eachone of the below-mentioned five enzymes at 30° C. for 60 minutes. Atthat time, the concentration of the wild-type enzyme and theconcentrations of the variant enzymes Y83F, Y83W, Y83L, and Y83N wererespectively 4×10⁻⁷ M, 8×10⁻⁷ M, 8×10⁻⁷ M, 8×10⁻⁶ M, and 8×10⁻⁶ M, inthis order. From each of the mixture solutions, 100-μl samples weresequentially taken out at intervals of 10 minutes; at each removal thesample's reaction was terminated with 30 μl of glacial acetic acid,followed by inactivation of the enzymes by 5 minutes of heating at 100°C. Subsequently, each sample was concentrated and dried. Then eachsample was dissolved in 5 μl of distilled water, and 2.5 μl of theresulting solution was spotted on a paper chromatography filter (size;25×40 cm, manufactured by Wattmann, Co., 3MM), for development at 55° C.for 2 hours in a pressure-resistant vessel containing a developmentsolvent (ethyl acetate: methanol: water=37:40:23). The development wasthen repeated, so that it was done twice. Subsequently, each of thefilters was dried and sprayed with 10 ml of 50 mM acetate buffer, pH 5.5containing 200 U Rhizopus glucoamylase; then the filter was kept in asealed vessel at 55° C. for 30 minutes. Then, on the filter, spotstreated with glucoamylase were detected by the silver nitrate coloringmethod. Coloring with silver nitrate was carried out as follows: Thefilter treated with glucoamylase was successively immersed in an acetonesolution of silver nitrate, an alcoholic sodium hydroxide solution, anda fixing solution, in this order; and it was successively dried. Thesubstrate and the enzymatic reaction products were assayedquantitatively, by measuring the coloring intensity of silvernitrate-colored spots on a paper chromatogram using a densitometer.

As a result, it was clearly shown that from the substrate G₇,transglycosylation products of longer chains: G₁₀, G₁₁, and G₁₂, wereprepared in all cases with each variant enzyme. Table 1 shows the ratioin % (conversion ratio in %) of transglycosylation reaction products(G≧₁₀) of a longer chain than maltodecaose (G₁₀) to the reduction of thesubstrate, when 40% of the substrate G₇ was consumed during theindividual enzymatic reactions. Compositional ratios (%) of G₁₀, G₁₁,and G₁₂ to the total G≧₁₀ products are also shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                 Rate of                                                                       conversion                                                                            Composition ratio in all                                              to G.sub.≧10                                                                   G.sub.≧10 (%)                                                  (%)     G.sub.10   G.sub.11                                                                             G.sub.12                                   ______________________________________                                        Wild-type   0        --         --   --                                       Y83F       65        25         55   20                                       Y83W       70        20         60   20                                       Y83L       50        25         55   20                                       Y83N       60        20         60   20                                       ______________________________________                                    

EXAMPLE 4

Transglycosylation activity of enzyme with Sfamy Y83 residue replaced

In order to analyze the transglycosylation activity of variant enzymes,enzyme reactions were carried out employing, as the substrate,maltopentaose with a p-nitrophenyl group introduced at its reducing endterminus (G₅ -PNP), and the products were quantitatively analyzed byHPLC (high-performance liquid chromatography). When 50% of G₅ -PNP wasconsumed, comparison was made of development patterns of reactionproducts produced by the wild-type enzyme and those of reaction productsproduced by each variant enzyme. With the wild-type enzyme, G₅ -PNP washydrolyzed principally into G₃ and G₂ -PNP, but there were notransglycosylation reaction products larger than G₅ -PNP. Alternatively,all of the variant enzymes produced p-nitrophenyl α-D-malto-octaoside(G₈ -PNP), which accounted for 50% of the products in total. Comparedwith the wild-type enzyme, the decomposition products; i.e. G₃ and G₂-PNP, in the reaction of each variant enzyme, were decreased to 30% and50%, respectively, of their initial amounts. This finding regarding thespecific G₃ reduction indicates that the G₃ prepared throughdecomposition is transferred onto the non-reducing end terminus of theunreacted substrate (G₅ -PNP), to prepare G₈ -PNP.

The occurrence of hydrolysis versus transglycosylation reaction dependson whether water molecules or substrate molecules attack, in anucleophilic manner, the carbonium ion intermediate as the enzymereaction intermediate. The obtained increase in the transglycosylatlonactivity in these variant enzymes can be explained by the reactionmechanism involving the carbonium ion intermediate. Based on the aboveexperimental evidence, it is demonstrated by the present inventors thatthe phenolic OH group of the Sfamy Y83 residue has a definitelyimportant function to provide H₂ O molecules to the carbonium ionintermediate. It has also been clearly shown that, in the variantenzymes, the function of the phenolic OH to provide H₂ O molecules isdeteriorated by the substitution of the Y83 residue with another aminoacid residue, so that the possibility of the otherwise unreactedsubstrate (in the presence of Y83 residue) attacking the carbonium ionintermediate, in a nucleophilic manner, is resultantly enhanced, toprepare transglycosylation reaction products of longer chains.

EXAMPLE 5

Hydrolysis activity of enzyme with Sfamy Y83 residue replaced

Furthermore, using a substrate with both its non-reducing end terminusand its reducing end terminus modified, i.e. 3KB-G₅ -CNP(chloronitrophenyl maltopentaose wherein the hydroxyl group at position3 of the glucose residue at the non-reducing end terminus isketobutylated; manufactured by Ono Pharmaceutical Company), thehydrolysis activities of the wild-type enzyme and the variant-typeenzymes were determined. The results are shown in Table 2. 3KB-G₅ -CNPcannot function as a substrate for transglycosylation reaction becauseof the modification of both of the termjul, and therefore, only thehydrolysis activity can be assayed.

                  TABLE 2                                                         ______________________________________                                        Wild-type    Y83F     Y83W     Y83L   Y83N                                    ______________________________________                                        kcat   9.2 × 10.sup.2                                                                    1.5 × 10.sup.2                                                                   1.3 × 10.sup.2                                                                 1.1 × 10.sup.1                                                                 1.6 × 10.sup.1                  (min.sup.-1)                                                                  Km (×                                                                          0.06      0.13     0.15   0.12   0.19                                  10.sup.-3 M)                                                                  kcat/Km                                                                              1.5 × 10.sup.7                                                                    1.2 × 10.sup.6                                                                   8.7 × 10.sup.5                                                                 9.3 × 10.sup.4                                                                 8.6 × 10.sup.4                  (min.sup.-1 ·                                                               (100%)    (8%)     (6%)   (1%)   (1%)                                  M.sup.-1)                                                                     ______________________________________                                    

The results shown in Table 2 indicate that in the variant-type enzymeY83F, in which the phenolic OH group was deleted from the Y83 residue,the hydrolysis activity (in kcat/Km) is reduced to one-tenth that of thewild-type enzyme, and that in the variant enzymes Y83L and Y83N, theactivity is reduced to one-hundredth that of the wild-type enzyme. Theseresults indicate that the phenolic OH group at the Y83 residue plays adefinitely important function to provide H₂ O molecules to the carboniumion intermediate.

EXAMPLE 6

Effects of replacing CGTaseY100 residue on transglycosylation activity

To each of two 100-ml portions of 50 mM acetate buffer, pH 5.5,containing 5% soluble starch and 5% sucrose, was added 700 THU(Tilden-Hudson unit) of one of wild-type CGTase and CGTaseY100W, therebypreparing respective enzyme mixtures, and reaction was allowed toproceed at 50° C. for 10 hours. Subsequently, the reaction mixtures werekept at 100° C. for 10 minutes, to inactivate each enzyme. The reactionproducts were concentrated and dried with a freeze-dryer. The driedproducts were then dissolved in 10 ml of distilled water, and theresulting samples were analyzed by paper chromatography and HPLC, todetermine the molecular species and sugar composition of the reactionproducts.

Paper chromatography was carried out as follows. An analytical samplesolution (2.5 μl ) was spotted on a paper chromatography paper(manufactured by Wattmann, Co., 3MM), of size 25×40 cm, for subsequentdevelopment at 55° C. for 2 hours in a pressure-resistant vesselcontaining a developing solvent (ethyl acetate: methanol:water=37:40:23). Then the development was repeated, so that it wasconducted twice. Subsequently, each of the filters was dried and sprayedwith 10 ml of 50 ntM acetate buffer, pH 5.5, containing 200 U Rhizopusglucoamylase; then the filter was kept in a sealed vessel at 55° C. for30 minutes. Then, on the filters, spots treated with glucoamylase weredetected by the previously described silver nitrate coloring method. Theenzymatic reaction products were quantitatively assayed, by measuringthe coloring intensity of silver nitrate-colored spots on a paperchromatogram using a densitometer. Consequently, five types ofoligosaccharides were detected in the spots of the reaction products ofboth of the enzymes.

By subsequently using the same method, preparative paper chromatographywas carried out to prepare the five types of oligosaccharides. The eachpurified oligosaccharide (200 μg) resultantly obtained was separatelydissolved in 20 μl of 50 mM acetate buffer, pH 5.5, followed by additionof 10 U glucoamylase; then it was kept at 50° C. for 20 minutes,followed by application to an MIC gel column (manufactured by MitsubishiChemicals, Co.) for liquid chromatography, to analyze the sugarcomposition at 65° C. The types of the transglcosylation reactionproducts produced by the wild-type enzyme reaction and by thevariant-type enzyme reaction (CGTaseY100W enzyme reaction), as well asthe compositional ratios of the products, are shown collectively inTable 3.

Table 3 shows that 50% of the initial sucrose in the CGTaseY100W enzymereaction was utilized as the receptor in the action oftransglycosylation, and that the resulting transglycosylation reactionproducts contained G₂ F (α-maltopyranoyl-β-D-fructofuranoside), G₃ F(α-maltotripyranoyl-β-D-fructofuranoside), and G≧₄ F (products of longerchains), at 21%, 12%, and 10%, respectively. In contrast, 25% of theinitial sucrose in the wild-type enzyme reaction was utilized as thereceptor in the action of transglycosylation, and the reaction productscontained G₂ F, G₃ F, and G≧₄ F at 9%, 6%, and 5%, respectively. Theseresults indicate that the transglycosylation activity of CGTaseY100W wasgreater than that of the wild-type enzyme.

                                      TABLE 3                                     __________________________________________________________________________    Rate of trans-                                                                              Composition of each sugar (%)                                   glycosylation*                                                                              Sucrose                                                                             Maltose                                                   (%)           (GF)  (G.sub.2)                                                                          G.sub.2 F                                                                         G.sub.3 F                                                                        G.sub.≧4 F                             __________________________________________________________________________    Wild-type                                                                           25      33    47    9   6  5                                            Y100W 50      24    33   21  12 10                                            __________________________________________________________________________     ##STR1##                                                                 

Having described our invention as related to the present embodiments, iti our intention that the invention not be limited by any of the detailsof the description, unless otherwise specified, but rather be construedbroadly within its spirit and scope as set out in the accompanyingclaims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 18                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1404 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Derived from plasmid pSfα1 (Agric. Biol. Chem.               (1985) 49:3089-3092)                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1404                                                         (D) OTHER INFORMATION: /note= "Nucleotides 1-1404                             correspond to nucleotides 79-1482 in the Saccharomycopsis                     fibuligera α- amylase structural gene"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAAACTAATGCTGATAAATGGAGATCACAGTCTATTTATCAAATTGTC48                            GluThrAsnAlaAspLysTrpArgSerGlnSerIleTyrGlnIleVal                              151015                                                                        ACTGACAGATTTGCTAGAACCGATGGTGATACAAGTGCTTCCTGTAAC96                            ThrAspArgPheAlaArgThrAspGlyAspThrSerAlaSerCysAsn                              202530                                                                        ACAGAAGATAGACTTTACTGTGGTGGTTCTTTCCAAGGCATCATAAAG144                           ThrGluAspArgLeuTyrCysGlyGlySerPheGlnGlyIleIleLys                              354045                                                                        AAGTTGGATTACATCAAAGATATGGGCTTTACTGCTATTTGGATTTCT192                           LysLeuAspTyrIleLysAspMetGlyPheThrAlaIleTrpIleSer                              505560                                                                        CCAGTTGTTGAAAACATTCCCGATAACACAGCATATGGTTATGCTTAT240                           ProValValGluAsnIleProAspAsnThrAlaTyrGlyTyrAlaTyr                              65707580                                                                      CATGGTTTCTGGATGAAGAACATATACAAAATTAATGAAAACTTTGGT288                           HisGlyPheTrpMetLysAsnIleTyrLysIleAsnGluAsnPheGly                              859095                                                                        ACTGCTGATGATTTGAAGTCTTTGGCACAAGAATTGCACGATCGTGAT336                           ThrAlaAspAspLeuLysSerLeuAlaGlnGluLeuHisAspArgAsp                              100105110                                                                     ATGTTGTTAATGGTGGATATCGTTACCAACCATTACGGCAGTGATGGC384                           MetLeuLeuMetValAspIleValThrAsnHisTyrGlySerAspGly                              115120125                                                                     AGTGGAGATAGTATCGATTACTCAGAGTACACCCCGTTCAACGACCAA432                           SerGlyAspSerIleAspTyrSerGluTyrThrProPheAsnAspGln                              130135140                                                                     AAGTACTTCCATAACTACTGTCTTATTTCAAACTATGATGACCAAGCT480                           LysTyrPheHisAsnTyrCysLeuIleSerAsnTyrAspAspGlnAla                              145150155160                                                                  CAGGTTCAAAGTTGCTGGGAAGGTGACTCTTCAGTTGCATTACCAGAT528                           GlnValGlnSerCysTrpGluGlyAspSerSerValAlaLeuProAsp                              165170175                                                                     TTGAGAACGGAAGATAGCGACGTGGCCTCAGTTTTCAATTCTTGGGTT576                           LeuArgThrGluAspSerAspValAlaSerValPheAsnSerTrpVal                              180185190                                                                     AAAGATTTTGTTGGCAATTACTCAATTGATGGTTTAAGAATTGATAGT624                           LysAspPheValGlyAsnTyrSerIleAspGlyLeuArgIleAspSer                              195200205                                                                     GCTAAACATGTGGACCAAGGCTTTTTCCCGGATTTTGTTAGTCCATCT672                           AlaLysHisValAspGlnGlyPhePheProAspPheValSerProSer                              210215220                                                                     GGAGTTTACTCAGTAGGCGAAGTTTTCCAAGGAGACCCAGCTTATACA720                           GlyValTyrSerValGlyGluValPheGlnGlyAspProAlaTyrThr                              225230235240                                                                  TGCCCATACCAAAATTACATTCCAGGGGTTAGTAATTATCCATTGTAC768                           CysProTyrGlnAsnTyrIleProGlyValSerAsnTyrProLeuTyr                              245250255                                                                     TACCCAACCACGAGATTTTTTAAAACTACTGATTCAAGTTCCAGTGAG816                           TyrProThrThrArgPhePheLysThrThrAspSerSerSerSerGlu                              260265270                                                                     TTGACTCAAATGATTTCAAGCGTTGCTTCCAGTTGTTCGGATCCAACT864                           LeuThrGlnMetIleSerSerValAlaSerSerCysSerAspProThr                              275280285                                                                     TTGTTGACAAACTTTGTAGAAAATCACGATAATGAAAGGTTCGCTTCA912                           LeuLeuThrAsnPheValGluAsnHisAspAsnGluArgPheAlaSer                              290295300                                                                     ATGACCAGCGACCAAAGTTTGATTTCTAATGCTATTGCATTTGTCCTT960                           MetThrSerAspGlnSerLeuIleSerAsnAlaIleAlaPheValLeu                              305310315320                                                                  TTGGGTGATGGTATTCCTGTCATTTACTATGGACAAGAACAAGGCTTG1008                          LeuGlyAspGlyIleProValIleTyrTyrGlyGlnGluGlnGlyLeu                              325330335                                                                     AGCGGAAAAAGTGACCCAAACAACAGAGAGGCCTTGTGGTTATCCGGC1056                          SerGlyLysSerAspProAsnAsnArgGluAlaLeuTrpLeuSerGly                              340345350                                                                     TACAACAAAGAGAGTGACTATTACAAGCTCATTGCCAAAGCTAATGCT1104                          TyrAsnLysGluSerAspTyrTyrLysLeuIleAlaLysAlaAsnAla                              355360365                                                                     GCCAGAAACGCCGCCGTTTATCAAGACTCAAGCTATGCCACCTCGCAG1152                          AlaArgAsnAlaAlaValTyrGlnAspSerSerTyrAlaThrSerGln                              370375380                                                                     CTTTCTGTGATCTTTTCAAATGACCATGTTATTGCAACAAAAAGAGGC1200                          LeuSerValIlePheSerAsnAspHisValIleAlaThrLysArgGly                              385390395400                                                                  AGCGTTGTTTCTGTTTTCAACAACCTTGGTTCCAGCGGTTCTTCTGAT1248                          SerValValSerValPheAsnAsnLeuGlySerSerGlySerSerAsp                              405410415                                                                     GTGACTATTTCCAACACAGGTTACAGTTCCGGTGAGGATTTGGTAGAA1296                          ValThrIleSerAsnThrGlyTyrSerSerGlyGluAspLeuValGlu                              420425430                                                                     GTTTTGACATGCAGTACTGTTAGCGGCAGCTCTGACTTACAAGTTTCT1344                          ValLeuThrCysSerThrValSerGlySerSerAspLeuGlnValSer                              435440445                                                                     ATCCAAGGTGGTCAACCACAAATCTTTGTTCCTGCTAAATATGCTTCT1392                          IleGlnGlyGlyGlnProGlnIlePheValProAlaLysTyrAlaSer                              450455460                                                                     GACATTTGTTCA1404                                                              AspIleCysSer                                                                  465                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 468 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GluThrAsnAlaAspLysTrpArgSerGlnSerIleTyrGlnIleVal                              151015                                                                        ThrAspArgPheAlaArgThrAspGlyAspThrSerAlaSerCysAsn                              202530                                                                        ThrGluAspArgLeuTyrCysGlyGlySerPheGlnGlyIleIleLys                              354045                                                                        LysLeuAspTyrIleLysAspMetGlyPheThrAlaIleTrpIleSer                              505560                                                                        ProValValGluAsnIleProAspAsnThrAlaTyrGlyTyrAlaTyr                              65707580                                                                      HisGlyPheTrpMetLysAsnIleTyrLysIleAsnGluAsnPheGly                              859095                                                                        ThrAlaAspAspLeuLysSerLeuAlaGlnGluLeuHisAspArgAsp                              100105110                                                                     MetLeuLeuMetValAspIleValThrAsnHisTyrGlySerAspGly                              115120125                                                                     SerGlyAspSerIleAspTyrSerGluTyrThrProPheAsnAspGln                              130135140                                                                     LysTyrPheHisAsnTyrCysLeuIleSerAsnTyrAspAspGlnAla                              145150155160                                                                  GlnValGlnSerCysTrpGluGlyAspSerSerValAlaLeuProAsp                              165170175                                                                     LeuArgThrGluAspSerAspValAlaSerValPheAsnSerTrpVal                              180185190                                                                     LysAspPheValGlyAsnTyrSerIleAspGlyLeuArgIleAspSer                              195200205                                                                     AlaLysHisValAspGlnGlyPhePheProAspPheValSerProSer                              210215220                                                                     GlyValTyrSerValGlyGluValPheGlnGlyAspProAlaTyrThr                              225230235240                                                                  CysProTyrGlnAsnTyrIleProGlyValSerAsnTyrProLeuTyr                              245250255                                                                     TyrProThrThrArgPhePheLysThrThrAspSerSerSerSerGlu                              260265270                                                                     LeuThrGlnMetIleSerSerValAlaSerSerCysSerAspProThr                              275280285                                                                     LeuLeuThrAsnPheValGluAsnHisAspAsnGluArgPheAlaSer                              290295300                                                                     MetThrSerAspGlnSerLeuIleSerAsnAlaIleAlaPheValLeu                              305310315320                                                                  LeuGlyAspGlyIleProValIleTyrTyrGlyGlnGluGlnGlyLeu                              325330335                                                                     SerGlyLysSerAspProAsnAsnArgGluAlaLeuTrpLeuSerGly                              340345350                                                                     TyrAsnLysGluSerAspTyrTyrLysLeuIleAlaLysAlaAsnAla                              355360365                                                                     AlaArgAsnAlaAlaValTyrGlnAspSerSerTyrAlaThrSerGln                              370375380                                                                     LeuSerValIlePheSerAsnAspHisValIleAlaThrLysArgGly                              385390395400                                                                  SerValValSerValPheAsnAsnLeuGlySerSerGlySerSerAsp                              405410415                                                                     ValThrIleSerAsnThrGlyTyrSerSerGlyGluAspLeuValGlu                              420425430                                                                     ValLeuThrCysSerThrValSerGlySerSerAspLeuGlnValSer                              435440445                                                                     IleGlnGlyGlyGlnProGlnIlePheValProAlaLysTyrAlaSer                              450455460                                                                     AspIleCysSer                                                                  465                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1404 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic nucleic acid"                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Derived from plasmid pSfα1 (Agric. Biol. Chem.               (1985) 49:3089-3092)                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1404                                                         (D) OTHER INFORMATION: /note= "Nucleotides 1-1404                             correspond to nucleotides 79-1482 in the Saccharomycopsis                     fibuligera α- amylase structural gene"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAAACTAATGCTGATAAATGGAGATCACAGTCTATTTATCAAATTGTC48                            GluThrAsnAlaAspLysTrpArgSerGlnSerIleTyrGlnIleVal                              151015                                                                        ACTGACAGATTTGCTAGAACCGATGGTGATACAAGTGCTTCCTGTAAC96                            ThrAspArgPheAlaArgThrAspGlyAspThrSerAlaSerCysAsn                              202530                                                                        ACAGAAGATAGACTTTACTGTGGTGGTTCTTTCCAAGGCATCATAAAG144                           ThrGluAspArgLeuTyrCysGlyGlySerPheGlnGlyIleIleLys                              354045                                                                        AAGTTGGATTACATCAAAGATATGGGCTTTACTGCTATTTGGATTTCT192                           LysLeuAspTyrIleLysAspMetGlyPheThrAlaIleTrpIleSer                              505560                                                                        CCAGTTGTTGAAAACATTCCCGATAACACAGCATATGGTTATGCTTAT240                           ProValValGluAsnIleProAspAsnThrAlaTyrGlyTyrAlaTyr                              65707580                                                                      CATGGTTGGTGGATGAAGAACATATACAAAATTAATGAAAACTTTGGT288                           HisGlyTrpTrpMetLysAsnIleTyrLysIleAsnGluAsnPheGly                              859095                                                                        ACTGCTGATGATTTGAAGTCTTTGGCACAAGAATTGCACGATCGTGAT336                           ThrAlaAspAspLeuLysSerLeuAlaGlnGluLeuHisAspArgAsp                              100105110                                                                     ATGTTGTTAATGGTGGATATCGTTACCAACCATTACGGCAGTGATGGC384                           MetLeuLeuMetValAspIleValThrAsnHisTyrGlySerAspGly                              115120125                                                                     AGTGGAGATAGTATCGATTACTCAGAGTACACCCCGTTCAACGACCAA432                           SerGlyAspSerIleAspTyrSerGluTyrThrProPheAsnAspGln                              130135140                                                                     AAGTACTTCCATAACTACTGTCTTATTTCAAACTATGATGACCAAGCT480                           LysTyrPheHisAsnTyrCysLeuIleSerAsnTyrAspAspGlnAla                              145150155160                                                                  CAGGTTCAAAGTTGCTGGGAAGGTGACTCTTCAGTTGCATTACCAGAT528                           GlnValGlnSerCysTrpGluGlyAspSerSerValAlaLeuProAsp                              165170175                                                                     TTGAGAACGGAAGATAGCGACGTGGCCTCAGTTTTCAATTCTTGGGTT576                           LeuArgThrGluAspSerAspValAlaSerValPheAsnSerTrpVal                              180185190                                                                     AAAGATTTTGTTGGCAATTACTCAATTGATGGTTTAAGAATTGATAGT624                           LysAspPheValGlyAsnTyrSerIleAspGlyLeuArgIleAspSer                              195200205                                                                     GCTAAACATGTGGACCAAGGCTTTTTCCCGGATTTTGTTAGTGCATCT672                           AlaLysHisValAspGlnGlyPhePheProAspPheValSerAlaSer                              210215220                                                                     GGAGTTTACTCAGTAGGCGAAGTTTTCCAAGGAGACCCAGCTTATACA720                           GlyValTyrSerValGlyGluValPheGlnGlyAspProAlaTyrThr                              225230235240                                                                  TGCCCATACCAAAATTACATTCCAGGGGTTAGTAATTATCCATTGTAC768                           CysProTyrGlnAsnTyrIleProGlyValSerAsnTyrProLeuTyr                              245250255                                                                     TACCCAACCACGAGATTTTTTAAAACTACTGATTCAAGTTCCAGTGAG816                           TyrProThrThrArgPhePheLysThrThrAspSerSerSerSerGlu                              260265270                                                                     TTGACTCAAATGATTTCAAGCGTTGCTTCCAGTTGTTCGGATCCAACT864                           LeuThrGlnMetIleSerSerValAlaSerSerCysSerAspProThr                              275280285                                                                     TTGTTGACAAACTTTGTAGAAAATCACGATAATGAAAGGTTCGCTTCA912                           LeuLeuThrAsnPheValGluAsnHisAspAsnGluArgPheAlaSer                              290295300                                                                     ATGACCAGCGACCAAAGTTTGATTTCTAATGCTATTGCATTTGTCCTT960                           MetThrSerAspGlnSerLeuIleSerAsnAlaIleAlaPheValLeu                              305310315320                                                                  TTGGGTGATGGTATTCCTGTCATTTACTATGGACAAGAACAAGGCTTG1008                          LeuGlyAspGlyIleProValIleTyrTyrGlyGlnGluGlnGlyLeu                              325330335                                                                     AGCGGAAAAAGTGACCCAAACAACAGAGAGGCCTTGTGGTTATCCGGC1056                          SerGlyLysSerAspProAsnAsnArgGluAlaLeuTrpLeuSerGly                              340345350                                                                     TACAACAAAGAGAGTGACTATTACAAGCTCATTGCCAAAGCTAATGCT1104                          TyrAsnLysGluSerAspTyrTyrLysLeuIleAlaLysAlaAsnAla                              355360365                                                                     GCCAGAAACGCCGCCGTTTATCAAGACTCAAGCTATGCCACCTCGCAG1152                          AlaArgAsnAlaAlaValTyrGlnAspSerSerTyrAlaThrSerGln                              370375380                                                                     CTTTCTGTGATCTTTTCAAATGACCATGTTATTGCAACAAAAAGAGGC1200                          LeuSerValIlePheSerAsnAspHisValIleAlaThrLysArgGly                              385390395400                                                                  AGCGTTGTTTCTGTTTTCAACAACCTTGGTTCCAGCGGTTCTTCTGAT1248                          SerValValSerValPheAsnAsnLeuGlySerSerGlySerSerAsp                              405410415                                                                     GTGACTATTTCCAACACAGGTTACAGTTCCGGTGAGGATTTGGTAGAA1296                          ValThrIleSerAsnThrGlyTyrSerSerGlyGluAspLeuValGlu                              420425430                                                                     GTTTTGACATGCAGTACTGTTAGCGGCAGCTCTGACTTACAAGTTTCT1344                          ValLeuThrCysSerThrValSerGlySerSerAspLeuGlnValSer                              435440445                                                                     ATCCAAGGTGGTCAACCACAAATCTTTGTTCCTGCTAAATATGCTTCT1392                          IleGlnGlyGlyGlnProGlnIlePheValProAlaLysTyrAlaSer                              450455460                                                                     GACATTTGTTCA1404                                                              AspIleCysSer                                                                  465                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 468 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GluThrAsnAlaAspLysTrpArgSerGlnSerIleTyrGlnIleVal                              151015                                                                        ThrAspArgPheAlaArgThrAspGlyAspThrSerAlaSerCysAsn                              202530                                                                        ThrGluAspArgLeuTyrCysGlyGlySerPheGlnGlyIleIleLys                              354045                                                                        LysLeuAspTyrIleLysAspMetGlyPheThrAlaIleTrpIleSer                              505560                                                                        ProValValGluAsnIleProAspAsnThrAlaTyrGlyTyrAlaTyr                              65707580                                                                      HisGlyTrpTrpMetLysAsnIleTyrLysIleAsnGluAsnPheGly                              859095                                                                        ThrAlaAspAspLeuLysSerLeuAlaGlnGluLeuHisAspArgAsp                              100105110                                                                     MetLeuLeuMetValAspIleValThrAsnHisTyrGlySerAspGly                              115120125                                                                     SerGlyAspSerIleAspTyrSerGluTyrThrProPheAsnAspGln                              130135140                                                                     LysTyrPheHisAsnTyrCysLeuIleSerAsnTyrAspAspGlnAla                              145150155160                                                                  GlnValGlnSerCysTrpGluGlyAspSerSerValAlaLeuProAsp                              165170175                                                                     LeuArgThrGluAspSerAspValAlaSerValPheAsnSerTrpVal                              180185190                                                                     LysAspPheValGlyAsnTyrSerIleAspGlyLeuArgIleAspSer                              195200205                                                                     AlaLysHisValAspGlnGlyPhePheProAspPheValSerAlaSer                              210215220                                                                     GlyValTyrSerValGlyGluValPheGlnGlyAspProAlaTyrThr                              225230235240                                                                  CysProTyrGlnAsnTyrIleProGlyValSerAsnTyrProLeuTyr                              245250255                                                                     TyrProThrThrArgPhePheLysThrThrAspSerSerSerSerGlu                              260265270                                                                     LeuThrGlnMetIleSerSerValAlaSerSerCysSerAspProThr                              275280285                                                                     LeuLeuThrAsnPheValGluAsnHisAspAsnGluArgPheAlaSer                              290295300                                                                     MetThrSerAspGlnSerLeuIleSerAsnAlaIleAlaPheValLeu                              305310315320                                                                  LeuGlyAspGlyIleProValIleTyrTyrGlyGlnGluGlnGlyLeu                              325330335                                                                     SerGlyLysSerAspProAsnAsnArgGluAlaLeuTrpLeuSerGly                              340345350                                                                     TyrAsnLysGluSerAspTyrTyrLysLeuIleAlaLysAlaAsnAla                              355360365                                                                     AlaArgAsnAlaAlaValTyrGlnAspSerSerTyrAlaThrSerGln                              370375380                                                                     LeuSerValIlePheSerAsnAspHisValIleAlaThrLysArgGly                              385390395400                                                                  SerValValSerValPheAsnAsnLeuGlySerSerGlySerSerAsp                              405410415                                                                     ValThrIleSerAsnThrGlyTyrSerSerGlyGluAspLeuValGlu                              420425430                                                                     ValLeuThrCysSerThrValSerGlySerSerAspLeuGlnValSer                              435440445                                                                     IleGlnGlyGlyGlnProGlnIlePheValProAlaLysTyrAlaSer                              450455460                                                                     AspIleCysSer                                                                  465                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1404 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Derived from plasmid pSfα1 (Agric. Biol. Chem.               (1985) 49:3089-3092)                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1404                                                         (D) OTHER INFORMATION: /note= "Nucleotides 1-1404                             correspond to nucleotides 79-1482 of the Saccharomycopis                      fibuligera α- amylase structural gene"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GAAACTAATGCTGATAAATGGAGATCACAGTCTATTTATCAAATTGTC48                            GluThrAsnAlaAspLysTrpArgSerGlnSerIleTyrGlnIleVal                              151015                                                                        ACTGACAGATTTGCTAGAACCGATGGTGATACAAGTGCTTCCTGTAAC96                            ThrAspArgPheAlaArgThrAspGlyAspThrSerAlaSerCysAsn                              202530                                                                        ACAGAAGATAGACTTTACTGTGGTGGTTCTTTCCAAGGCATCATAAAG144                           ThrGluAspArgLeuTyrCysGlyGlySerPheGlnGlyIleIleLys                              354045                                                                        AAGTTGGATTACATCAAAGATATGGGCTTTACTGCTATTTGGATTTCT192                           LysLeuAspTyrIleLysAspMetGlyPheThrAlaIleTrpIleSer                              505560                                                                        CCAGTTGTTGAAAACATTCCCGATAACACAGCATATGGTTATGCTTAT240                           ProValValGluAsnIleProAspAsnThrAlaTyrGlyTyrAlaTyr                              65707580                                                                      CATGGTCTCTGGATGAAGAACATATACAAAATTAATGAAAACTTTGGT288                           HisGlyLeuTrpMetLysAsnIleTyrLysIleAsnGluAsnPheGly                              859095                                                                        ACTGCTGATGATTTGAAGTCTTTGGCACAAGAATTGCACGATCGTGAT336                           ThrAlaAspAspLeuLysSerLeuAlaGlnGluLeuHisAspArgAsp                              100105110                                                                     ATGTTGTTAATGGTGGATATCGTTACCAACCATTACGGCAGTGATGGC384                           MetLeuLeuMetValAspIleValThrAsnHisTyrGlySerAspGly                              115120125                                                                     AGTGGAGATAGTATCGATTACTCAGAGTACACCCCGTTCAACGACCAA432                           SerGlyAspSerIleAspTyrSerGluTyrThrProPheAsnAspGln                              130135140                                                                     AAGTACTTCCATAACTACTGTCTTATTTCAAACTATGATGACCAAGCT480                           LysTyrPheHisAsnTyrCysLeuIleSerAsnTyrAspAspGlnAla                              145150155160                                                                  CAGGTTCAAAGTTGCTGGGAAGGTGACTCTTCAGTTGCATTACCAGAT528                           GlnValGlnSerCysTrpGluGlyAspSerSerValAlaLeuProAsp                              165170175                                                                     TTGAGAACGGAAGATAGCGACGTGGCCTCAGTTTTCAATTCTTGGGTT576                           LeuArgThrGluAspSerAspValAlaSerValPheAsnSerTrpVal                              180185190                                                                     AAAGATTTTGTTGGCAATTACTCAATTGATGGTTTAAGAATTGATAGT624                           LysAspPheValGlyAsnTyrSerIleAspGlyLeuArgIleAspSer                              195200205                                                                     GCTAAACATGTGGACCAAGGCTTTTTCCCGGATTTTGTTAGTGCATCT672                           AlaLysHisValAspGlnGlyPhePheProAspPheValSerAlaSer                              210215220                                                                     GGAGTTTACTCAGTAGGCGAAGTTTTCCAAGGAGACCCAGCTTATACA720                           GlyValTyrSerValGlyGluValPheGlnGlyAspProAlaTyrThr                              225230235240                                                                  TGCCCATACCAAAATTACATTCCAGGGGTTAGTAATTATCCATTGTAC768                           CysProTyrGlnAsnTyrIleProGlyValSerAsnTyrProLeuTyr                              245250255                                                                     TACCCAACCACGAGATTTTTTAAAACTACTGATTCAAGTTCCAGTGAG816                           TyrProThrThrArgPhePheLysThrThrAspSerSerSerSerGlu                              260265270                                                                     TTGACTCAAATGATTTCAAGCGTTGCTTCCAGTTGTTCGGATCCAACT864                           LeuThrGlnMetIleSerSerValAlaSerSerCysSerAspProThr                              275280285                                                                     TTGTTGACAAACTTTGTAGAAAATCACGATAATGAAAGGTTCGCTTCA912                           LeuLeuThrAsnPheValGluAsnHisAspAsnGluArgPheAlaSer                              290295300                                                                     ATGACCAGCGACCAAAGTTTGATTTCTAATGCTATTGCATTTGTCCTT960                           MetThrSerAspGlnSerLeuIleSerAsnAlaIleAlaPheValLeu                              305310315320                                                                  TTGGGTGATGGTATTCCTGTCATTTACTATGGACAAGAACAAGGCTTG1008                          LeuGlyAspGlyIleProValIleTyrTyrGlyGlnGluGlnGlyLeu                              325330335                                                                     AGCGGAAAAAGTGACCCAAACAACAGAGAGGCCTTGTGGTTATCCGGC1056                          SerGlyLysSerAspProAsnAsnArgGluAlaLeuTrpLeuSerGly                              340345350                                                                     TACAACAAAGAGAGTGACTATTACAAGCTCATTGCCAAAGCTAATGCT1104                          TyrAsnLysGluSerAspTyrTyrLysLeuIleAlaLysAlaAsnAla                              355360365                                                                     GCCAGAAACGCCGCCGTTTATCAAGACTCAAGCTATGCCACCTCGCAG1152                          AlaArgAsnAlaAlaValTyrGlnAspSerSerTyrAlaThrSerGln                              370375380                                                                     CTTTCTGTGATCTTTTCAAATGACCATGTTATTGCAACAAAAAGAGGC1200                          LeuSerValIlePheSerAsnAspHisValIleAlaThrLysArgGly                              385390395400                                                                  AGCGTTGTTTCTGTTTTCAACAACCTTGGTTCCAGCGGTTCTTCTGAT1248                          SerValValSerValPheAsnAsnLeuGlySerSerGlySerSerAsp                              405410415                                                                     GTGACTATTTCCAACACAGGTTACAGTTCCGGTGAGGATTTGGTAGAA1296                          ValThrIleSerAsnThrGlyTyrSerSerGlyGluAspLeuValGlu                              420425430                                                                     GTTTTGACATGCAGTACTGTTAGCGGCAGCTCTGACTTACAAGTTTCT1344                          ValLeuThrCysSerThrValSerGlySerSerAspLeuGlnValSer                              435440445                                                                     ATCCAAGGTGGTCAACCACAAATCTTTGTTCCTGCTAAATATGCTTCT1392                          IleGlnGlyGlyGlnProGlnIlePheValProAlaLysTyrAlaSer                              450455460                                                                     GACATTTGTTCA1404                                                              AspIleCysSer                                                                  465                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 468 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GluThrAsnAlaAspLysTrpArgSerGlnSerIleTyrGlnIleVal                              151015                                                                        ThrAspArgPheAlaArgThrAspGlyAspThrSerAlaSerCysAsn                              202530                                                                        ThrGluAspArgLeuTyrCysGlyGlySerPheGlnGlyIleIleLys                              354045                                                                        LysLeuAspTyrIleLysAspMetGlyPheThrAlaIleTrpIleSer                              505560                                                                        ProValValGluAsnIleProAspAsnThrAlaTyrGlyTyrAlaTyr                              65707580                                                                      HisGlyLeuTrpMetLysAsnIleTyrLysIleAsnGluAsnPheGly                              859095                                                                        ThrAlaAspAspLeuLysSerLeuAlaGlnGluLeuHisAspArgAsp                              100105110                                                                     MetLeuLeuMetValAspIleValThrAsnHisTyrGlySerAspGly                              115120125                                                                     SerGlyAspSerIleAspTyrSerGluTyrThrProPheAsnAspGln                              130135140                                                                     LysTyrPheHisAsnTyrCysLeuIleSerAsnTyrAspAspGlnAla                              145150155160                                                                  GlnValGlnSerCysTrpGluGlyAspSerSerValAlaLeuProAsp                              165170175                                                                     LeuArgThrGluAspSerAspValAlaSerValPheAsnSerTrpVal                              180185190                                                                     LysAspPheValGlyAsnTyrSerIleAspGlyLeuArgIleAspSer                              195200205                                                                     AlaLysHisValAspGlnGlyPhePheProAspPheValSerAlaSer                              210215220                                                                     GlyValTyrSerValGlyGluValPheGlnGlyAspProAlaTyrThr                              225230235240                                                                  CysProTyrGlnAsnTyrIleProGlyValSerAsnTyrProLeuTyr                              245250255                                                                     TyrProThrThrArgPhePheLysThrThrAspSerSerSerSerGlu                              260265270                                                                     LeuThrGlnMetIleSerSerValAlaSerSerCysSerAspProThr                              275280285                                                                     LeuLeuThrAsnPheValGluAsnHisAspAsnGluArgPheAlaSer                              290295300                                                                     MetThrSerAspGlnSerLeuIleSerAsnAlaIleAlaPheValLeu                              305310315320                                                                  LeuGlyAspGlyIleProValIleTyrTyrGlyGlnGluGlnGlyLeu                              325330335                                                                     SerGlyLysSerAspProAsnAsnArgGluAlaLeuTrpLeuSerGly                              340345350                                                                     TyrAsnLysGluSerAspTyrTyrLysLeuIleAlaLysAlaAsnAla                              355360365                                                                     AlaArgAsnAlaAlaValTyrGlnAspSerSerTyrAlaThrSerGln                              370375380                                                                     LeuSerValIlePheSerAsnAspHisValIleAlaThrLysArgGly                              385390395400                                                                  SerValValSerValPheAsnAsnLeuGlySerSerGlySerSerAsp                              405410415                                                                     ValThrIleSerAsnThrGlyTyrSerSerGlyGluAspLeuValGlu                              420425430                                                                     ValLeuThrCysSerThrValSerGlySerSerAspLeuGlnValSer                              435440445                                                                     IleGlnGlyGlyGlnProGlnIlePheValProAlaLysTyrAlaSer                              450455460                                                                     AspIleCysSer                                                                  465                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1404 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Derived from plasmid pSfα1 (Agric. Biol. Chem.               (1985) 49:3089-3092)                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1404                                                         (D) OTHER INFORMATION: /note= "Nucleotides 1-1404                             correspond to nucleotides 79-1482 of the Saccharomycopsis                     fibuligera α- amylase structural gene"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GAAACTAATGCTGATAAATGGAGATCACAGTCTATTTATCAAATTGTC48                            GluThrAsnAlaAspLysTrpArgSerGlnSerIleTyrGlnIleVal                              151015                                                                        ACTGACAGATTTGCTAGAACCGATGGTGATACAAGTGCTTCCTGTAAC96                            ThrAspArgPheAlaArgThrAspGlyAspThrSerAlaSerCysAsn                              202530                                                                        ACAGAAGATAGACTTTACTGTGGTGGTTCTTTCCAAGGCATCATAAAG144                           ThrGluAspArgLeuTyrCysGlyGlySerPheGlnGlyIleIleLys                              354045                                                                        AAGTTGGATTACATCAAAGATATGGGCTTTACTGCTATTTGGATTTCT192                           LysLeuAspTyrIleLysAspMetGlyPheThrAlaIleTrpIleSer                              505560                                                                        CCAGTTGTTGAAAACATTCCCGATAACACAGCATATGGTTATGCTTAT240                           ProValValGluAsnIleProAspAsnThrAlaTyrGlyTyrAlaTyr                              65707580                                                                      CATGGTAACTGGATGAAGAACATATACAAAATTAATGAAAACTTTGGT288                           HisGlyAsnTrpMetLysAsnIleTyrLysIleAsnGluAsnPheGly                              859095                                                                        ACTGCTGATGATTTGAAGTCTTTGGCACAAGAATTGCACGATCGTGAT336                           ThrAlaAspAspLeuLysSerLeuAlaGlnGluLeuHisAspArgAsp                              100105110                                                                     ATGTTGTTAATGGTGGATATCGTTACCAACCATTACGGCAGTGATGGC384                           MetLeuLeuMetValAspIleValThrAsnHisTyrGlySerAspGly                              115120125                                                                     AGTGGAGATAGTATCGATTACTCAGAGTACACCCCGTTCAACGACCAA432                           SerGlyAspSerIleAspTyrSerGluTyrThrProPheAsnAspGln                              130135140                                                                     AAGTACTTCCATAACTACTGTCTTATTTCAAACTATGATGACCAAGCT480                           LysTyrPheHisAsnTyrCysLeuIleSerAsnTyrAspAspGlnAla                              145150155160                                                                  CAGGTTCAAAGTTGCTGGGAAGGTGACTCTTCAGTTGCATTACCAGAT528                           GlnValGlnSerCysTrpGluGlyAspSerSerValAlaLeuProAsp                              165170175                                                                     TTGAGAACGGAAGATAGCGACGTGGCCTCAGTTTTCAATTCTTGGGTT576                           LeuArgThrGluAspSerAspValAlaSerValPheAsnSerTrpVal                              180185190                                                                     AAAGATTTTGTTGGCAATTACTCAATTGATGGTTTAAGAATTGATAGT624                           LysAspPheValGlyAsnTyrSerIleAspGlyLeuArgIleAspSer                              195200205                                                                     GCTAAACATGTGGACCAAGGCTTTTTCCCGGATTTTGTTAGTGCATCT672                           AlaLysHisValAspGlnGlyPhePheProAspPheValSerAlaSer                              210215220                                                                     GGAGTTTACTCAGTAGGCGAAGTTTTCCAAGGAGACCCAGCTTATACA720                           GlyValTyrSerValGlyGluValPheGlnGlyAspProAlaTyrThr                              225230235240                                                                  TGCCCATACCAAAATTACATTCCAGGGGTTAGTAATTATCCATTGTAC768                           CysProTyrGlnAsnTyrIleProGlyValSerAsnTyrProLeuTyr                              245250255                                                                     TACCCAACCACGAGATTTTTTAAAACTACTGATTCAAGTTCCAGTGAG816                           TyrProThrThrArgPhePheLysThrThrAspSerSerSerSerGlu                              260265270                                                                     TTGACTCAAATGATTTCAAGCGTTGCTTCCAGTTGTTCGGATCCAACT864                           LeuThrGlnMetIleSerSerValAlaSerSerCysSerAspProThr                              275280285                                                                     TTGTTGACAAACTTTGTAGAAAATCACGATAATGAAAGGTTCGCTTCA912                           LeuLeuThrAsnPheValGluAsnHisAspAsnGluArgPheAlaSer                              290295300                                                                     ATGACCAGCGACCAAAGTTTGATTTCTAATGCTATTGCATTTGTCCTT960                           MetThrSerAspGlnSerLeuIleSerAsnAlaIleAlaPheValLeu                              305310315320                                                                  TTGGGTGATGGTATTCCTGTCATTTACTATGGACAAGAACAAGGCTTG1008                          LeuGlyAspGlyIleProValIleTyrTyrGlyGlnGluGlnGlyLeu                              325330335                                                                     AGCGGAAAAAGTGACCCAAACAACAGAGAGGCCTTGTGGTTATCCGGC1056                          SerGlyLysSerAspProAsnAsnArgGluAlaLeuTrpLeuSerGly                              340345350                                                                     TACAACAAAGAGAGTGACTATTACAAGCTCATTGCCAAAGCTAATGCT1104                          TyrAsnLysGluSerAspTyrTyrLysLeuIleAlaLysAlaAsnAla                              355360365                                                                     GCCAGAAACGCCGCCGTTTATCAAGACTCAAGCTATGCCACCTCGCAG1152                          AlaArgAsnAlaAlaValTyrGlnAspSerSerTyrAlaThrSerGln                              370375380                                                                     CTTTCTGTGATCTTTTCAAATGACCATGTTATTGCAACAAAAAGAGGC1200                          LeuSerValIlePheSerAsnAspHisValIleAlaThrLysArgGly                              385390395400                                                                  AGCGTTGTTTCTGTTTTCAACAACCTTGGTTCCAGCGGTTCTTCTGAT1248                          SerValValSerValPheAsnAsnLeuGlySerSerGlySerSerAsp                              405410415                                                                     GTGACTATTTCCAACACAGGTTACAGTTCCGGTGAGGATTTGGTAGAA1296                          ValThrIleSerAsnThrGlyTyrSerSerGlyGluAspLeuValGlu                              420425430                                                                     GTTTTGACATGCAGTACTGTTAGCGGCAGCTCTGACTTACAAGTTTCT1344                          ValLeuThrCysSerThrValSerGlySerSerAspLeuGlnValSer                              435440445                                                                     ATCCAAGGTGGTCAACCACAAATCTTTGTTCCTGCTAAATATGCTTCT1392                          IleGlnGlyGlyGlnProGlnIlePheValProAlaLysTyrAlaSer                              450455460                                                                     GACATTTGTTCA1404                                                              AspIleCysSer                                                                  465                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 468 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GluThrAsnAlaAspLysTrpArgSerGlnSerIleTyrGlnIleVal                              151015                                                                        ThrAspArgPheAlaArgThrAspGlyAspThrSerAlaSerCysAsn                              202530                                                                        ThrGluAspArgLeuTyrCysGlyGlySerPheGlnGlyIleIleLys                              354045                                                                        LysLeuAspTyrIleLysAspMetGlyPheThrAlaIleTrpIleSer                              505560                                                                        ProValValGluAsnIleProAspAsnThrAlaTyrGlyTyrAlaTyr                              65707580                                                                      HisGlyAsnTrpMetLysAsnIleTyrLysIleAsnGluAsnPheGly                              859095                                                                        ThrAlaAspAspLeuLysSerLeuAlaGlnGluLeuHisAspArgAsp                              100105110                                                                     MetLeuLeuMetValAspIleValThrAsnHisTyrGlySerAspGly                              115120125                                                                     SerGlyAspSerIleAspTyrSerGluTyrThrProPheAsnAspGln                              130135140                                                                     LysTyrPheHisAsnTyrCysLeuIleSerAsnTyrAspAspGlnAla                              145150155160                                                                  GlnValGlnSerCysTrpGluGlyAspSerSerValAlaLeuProAsp                              165170175                                                                     LeuArgThrGluAspSerAspValAlaSerValPheAsnSerTrpVal                              180185190                                                                     LysAspPheValGlyAsnTyrSerIleAspGlyLeuArgIleAspSer                              195200205                                                                     AlaLysHisValAspGlnGlyPhePheProAspPheValSerAlaSer                              210215220                                                                     GlyValTyrSerValGlyGluValPheGlnGlyAspProAlaTyrThr                              225230235240                                                                  CysProTyrGlnAsnTyrIleProGlyValSerAsnTyrProLeuTyr                              245250255                                                                     TyrProThrThrArgPhePheLysThrThrAspSerSerSerSerGlu                              260265270                                                                     LeuThrGlnMetIleSerSerValAlaSerSerCysSerAspProThr                              275280285                                                                     LeuLeuThrAsnPheValGluAsnHisAspAsnGluArgPheAlaSer                              290295300                                                                     MetThrSerAspGlnSerLeuIleSerAsnAlaIleAlaPheValLeu                              305310315320                                                                  LeuGlyAspGlyIleProValIleTyrTyrGlyGlnGluGlnGlyLeu                              325330335                                                                     SerGlyLysSerAspProAsnAsnArgGluAlaLeuTrpLeuSerGly                              340345350                                                                     TyrAsnLysGluSerAspTyrTyrLysLeuIleAlaLysAlaAsnAla                              355360365                                                                     AlaArgAsnAlaAlaValTyrGlnAspSerSerTyrAlaThrSerGln                              370375380                                                                     LeuSerValIlePheSerAsnAspHisValIleAlaThrLysArgGly                              385390395400                                                                  SerValValSerValPheAsnAsnLeuGlySerSerGlySerSerAsp                              405410415                                                                     ValThrIleSerAsnThrGlyTyrSerSerGlyGluAspLeuValGlu                              420425430                                                                     ValLeuThrCysSerThrValSerGlySerSerAspLeuGlnValSer                              435440445                                                                     IleGlnGlyGlyGlnProGlnIlePheValProAlaLysTyrAlaSer                              450455460                                                                     AspIleCysSer                                                                  465                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2061 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: Library of chromosomal DNA from Bacillus                         macerans, pMAC, generated by treating chromosomal DNA                         from Bacillus macerans IAM1243 with a restriction enzyme,                     and inserting and linking restriction fragments to pBR322                     (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..2061                                                         (D) OTHER INFORMATION: /note= "Nucleotides 1-2061                             correspond to nucleotides 82-2142 of the Bacillus                             macerans cyclomaltodextrin glucanotransferase structural                      gene"                                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCACCCGATACGAGCGTGGACAACAAGGTCAATTTCAGTACGGACGTC48                            SerProAspThrSerValAspAsnLysValAsnPheSerThrAspVal                              151015                                                                        ATCTATCAGATTGTGACCGACCGCTTCGCGGACGGGGACAGGACGAAC96                            IleTyrGlnIleValThrAspArgPheAlaAspGlyAspArgThrAsn                              202530                                                                        AATCCGGCGGGGGATGCGTTCAGCGGCGACCGATCCAATTTGAAGCTC144                           AsnProAlaGlyAspAlaPheSerGlyAspArgSerAsnLeuLysLeu                              354045                                                                        TATTTCGGGGGAGACTGGCAGGGGATTATCGACAAGATTAACGACGGT192                           TyrPheGlyGlyAspTrpGlnGlyIleIleAspLysIleAsnAspGly                              505560                                                                        TATTTGACCGGCATGGGCGTCACCGCCCTCTGGATATCCCAACCTGTG240                           TyrLeuThrGlyMetGlyValThrAlaLeuTrpIleSerGlnProVal                              65707580                                                                      GAAAATATCACCTCCGTCATCAAGTATTCCGGCGTTAACAATACGTCT288                           GluAsnIleThrSerValIleLysTyrSerGlyValAsnAsnThrSer                              859095                                                                        TATCACGGTTGGTGGGCGAGGGATTTTAAGCAAACCAACGACGCTTTC336                           TyrHisGlyTrpTrpAlaArgAspPheLysGlnThrAsnAspAlaPhe                              100105110                                                                     GGGGATTTTGCCGATTTTCAAAATCTGATTGATACGCTCACGCTCATA384                           GlyAspPheAlaAspPheGlnAsnLeuIleAspThrLeuThrLeuIle                              115120125                                                                     ACATCAAGGTCGGATCGACTTCGCCCCCAACCACACGTCTCCGGCCGA432                           ThrSerArgSerAspArgLeuArgProGlnProHisValSerGlyArg                              130135140                                                                     GCAGGGACGAACCCCGGCTTCGCCGAGAACGGTGCGCTGTATGATAAC480                           AlaGlyThrAsnProGlyPheAlaGluAsnGlyAlaLeuTyrAspAsn                              145150155160                                                                  GGTTCGCTGCTCGGCGCCTACAGCAATGATACGGCCGGCCTTTTCCAT528                           GlySerLeuLeuGlyAlaTyrSerAsnAspThrAlaGlyLeuPheHis                              165170175                                                                     CATAACGGGGGGACCGATTTTTCCACGATTGAAGACGGTATTTACAAG576                           HisAsnGlyGlyThrAspPheSerThrIleGluAspGlyIleTyrLys                              180185190                                                                     AACCTCTACGACCTGGCGGACATCAACCATAACAACAACGCTATGGAC624                           AsnLeuTyrAspLeuAlaAspIleAsnHisAsnAsnAsnAlaMetAsp                              195200205                                                                     GCTTATTTTAAAAGCGCTATCGACCTTTGGCTCGGCATGGGTGTGGAC672                           AlaTyrPheLysSerAlaIleAspLeuTrpLeuGlyMetGlyValAsp                              210215220                                                                     GGGATTCGTTTTGACGGGGTGAAGCAGTATCCTTTCGGCTGGCAAAAA720                           GlyIleArgPheAspGlyValLysGlnTyrProPheGlyTrpGlnLys                              225230235240                                                                  AGCTTCGTTTCCTCGATTTACGGCGGCGATCATCCGGTATTTACGTTC768                           SerPheValSerSerIleTyrGlyGlyAspHisProValPheThrPhe                              245250255                                                                     GGGGAATGGTATCTTGGCGCGGATCAAACCGACGGAGACAACATTAAA816                           GlyGluTrpTyrLeuGlyAlaAspGlnThrAspGlyAspAsnIleLys                              260265270                                                                     TTCGCCAACGAAAGCGGGATGAACCTGCTGGACTTTGAATACGCGCAG864                           PheAlaAsnGluSerGlyMetAsnLeuLeuAspPheGluTyrAlaGln                              275280285                                                                     GAAGTGCGCGAAGTGTTCCGGGACAAAACGGAAACGATGAAGGATCTC912                           GluValArgGluValPheArgAspLysThrGluThrMetLysAspLeu                              290295300                                                                     TATGAGGTGCTGGCCAGCACGGAGTCGCAATACGACTACATCAACAAT960                           TyrGluValLeuAlaSerThrGluSerGlnTyrAspTyrIleAsnAsn                              305310315320                                                                  ATGGTGACCTTCATCGACAACCATGATATGGACCGGTTCCAGGTTGCC1008                          MetValThrPheIleAspAsnHisAspMetAspArgPheGlnValAla                              325330335                                                                     GGTTCCGGTACGCGGGCGACCGAGCAAGCGTTGGCGCTGACGCTGACT1056                          GlySerGlyThrArgAlaThrGluGlnAlaLeuAlaLeuThrLeuThr                              340345350                                                                     TCCCGCGGCGTGCCAGCCATCTACTACGGCACGGAGCAGTACATGACC1104                          SerArgGlyValProAlaIleTyrTyrGlyThrGluGlnTyrMetThr                              355360365                                                                     GGCGATGGCGACCCCAACAACCGGGCGATGATGACCTCGTTTAATACC1152                          GlyAspGlyAspProAsnAsnArgAlaMetMetThrSerPheAsnThr                              370375380                                                                     GGGACGACGGCTTATAAAGTGATTCAGGCATTGGCGCCGCTGCGTAAA1200                          GlyThrThrAlaTyrLysValIleGlnAlaLeuAlaProLeuArgLys                              385390395400                                                                  TCCAATCCGGCCATCGCTTATGGGACGACGACAGAGCGCTGGGTTAAC1248                          SerAsnProAlaIleAlaTyrGlyThrThrThrGluArgTrpValAsn                              405410415                                                                     AACGATGTGTTGATTATTGAACGCAAATTCGGCAGCAGCGCCGCTTTG1296                          AsnAspValLeuIleIleGluArgLysPheGlySerSerAlaAlaLeu                              420425430                                                                     GTGGCGATTAATCGAAACTCGTCCGCCGCTTATCCGATTTCGGGTCTG1344                          ValAlaIleAsnArgAsnSerSerAlaAlaTyrProIleSerGlyLeu                              435440445                                                                     TTGAGTTCGCTGCCGGCGGGCACTTATTCGGATGTATTGAACGGACTC1392                          LeuSerSerLeuProAlaGlyThrTyrSerAspValLeuAsnGlyLeu                              450455460                                                                     TTAAACGGCAACTCCATTACCGTGGGCAGCGGCGGCGCCGTCACCAAC1440                          LeuAsnGlyAsnSerIleThrValGlySerGlyGlyAlaValThrAsn                              465470475480                                                                  TTTACGCTGGCGGCCGGCGGCACGGCGGTATGGCAGTACACAGCGCCG1488                          PheThrLeuAlaAlaGlyGlyThrAlaValTrpGlnTyrThrAlaPro                              485490495                                                                     GAAACGTCGCCGGCGATCGGCAATGTGGGTCCCACCATGGGCCAGCCG1536                          GluThrSerProAlaIleGlyAsnValGlyProThrMetGlyGlnPro                              500505510                                                                     GGGAATATAGTGACGATTGACGGCCGCGGCTTTGGCGGCACGGCGGGC1584                          GlyAsnIleValThrIleAspGlyArgGlyPheGlyGlyThrAlaGly                              515520525                                                                     ACGGTTTATTTCGGGACGACGGCGGTGACCGGCTCCGGCATCGTAAGC1632                          ThrValTyrPheGlyThrThrAlaValThrGlySerGlyIleValSer                              530535540                                                                     TGGGAGGACACGCAGATTAAGGCGGTCATACCGAAGGTCGCGGCGGGC1680                          TrpGluAspThrGlnIleLysAlaValIleProLysValAlaAlaGly                              545550555560                                                                  AAAACGGGCGTATCGGTCAAAACGTCGTCCGGCACCGCCAGCAATACA1728                          LysThrGlyValSerValLysThrSerSerGlyThrAlaSerAsnThr                              565570575                                                                     TTCAAAAGCTTCAATGTACTGACGGGGGATCAGGTCACGGTGCGTTTC1776                          PheLysSerPheAsnValLeuThrGlyAspGlnValThrValArgPhe                              580585590                                                                     CTGGTCAATCAAGCCAATACCAATTACGGAACAAATGTTTATCTTGTC1824                          LeuValAsnGlnAlaAsnThrAsnTyrGlyThrAsnValTyrLeuVal                              595600605                                                                     GGCAACGCCGCCGAGCTCGGCACCTGGGACCCGAACAAAGCGATTGGG1872                          GlyAsnAlaAlaGluLeuGlyThrTrpAspProAsnLysAlaIleGly                              610615620                                                                     CCGATGTACAATCAGGTGATCGCCAAGTACCCGTCCTGGTATTACGAT1920                          ProMetTyrAsnGlnValIleAlaLysTyrProSerTrpTyrTyrAsp                              625630635640                                                                  GTCAGCGTGCCGGCGGGGACAAAGCTGGATTTTAAATTTATTAAAAAG1968                          ValSerValProAlaGlyThrLysLeuAspPheLysPheIleLysLys                              645650655                                                                     GGCGGCGGTACGGTGACTTGGGAAGGCGGGGGCAACCATACGTACACG2016                          GlyGlyGlyThrValThrTrpGluGlyGlyGlyAsnHisThrTyrThr                              660665670                                                                     ACGCCGGCCAGCGGCGTAGGGACGGTGACGGTGGACTGGCAAAAT2061                             ThrProAlaSerGlyValGlyThrValThrValAspTrpGlnAsn                                 675680685                                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 687 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      SerProAspThrSerValAspAsnLysValAsnPheSerThrAspVal                              151015                                                                        IleTyrGlnIleValThrAspArgPheAlaAspGlyAspArgThrAsn                              202530                                                                        AsnProAlaGlyAspAlaPheSerGlyAspArgSerAsnLeuLysLeu                              354045                                                                        TyrPheGlyGlyAspTrpGlnGlyIleIleAspLysIleAsnAspGly                              505560                                                                        TyrLeuThrGlyMetGlyValThrAlaLeuTrpIleSerGlnProVal                              65707580                                                                      GluAsnIleThrSerValIleLysTyrSerGlyValAsnAsnThrSer                              859095                                                                        TyrHisGlyTrpTrpAlaArgAspPheLysGlnThrAsnAspAlaPhe                              100105110                                                                     GlyAspPheAlaAspPheGlnAsnLeuIleAspThrLeuThrLeuIle                              115120125                                                                     ThrSerArgSerAspArgLeuArgProGlnProHisValSerGlyArg                              130135140                                                                     AlaGlyThrAsnProGlyPheAlaGluAsnGlyAlaLeuTyrAspAsn                              145150155160                                                                  GlySerLeuLeuGlyAlaTyrSerAsnAspThrAlaGlyLeuPheHis                              165170175                                                                     HisAsnGlyGlyThrAspPheSerThrIleGluAspGlyIleTyrLys                              180185190                                                                     AsnLeuTyrAspLeuAlaAspIleAsnHisAsnAsnAsnAlaMetAsp                              195200205                                                                     AlaTyrPheLysSerAlaIleAspLeuTrpLeuGlyMetGlyValAsp                              210215220                                                                     GlyIleArgPheAspGlyValLysGlnTyrProPheGlyTrpGlnLys                              225230235240                                                                  SerPheValSerSerIleTyrGlyGlyAspHisProValPheThrPhe                              245250255                                                                     GlyGluTrpTyrLeuGlyAlaAspGlnThrAspGlyAspAsnIleLys                              260265270                                                                     PheAlaAsnGluSerGlyMetAsnLeuLeuAspPheGluTyrAlaGln                              275280285                                                                     GluValArgGluValPheArgAspLysThrGluThrMetLysAspLeu                              290295300                                                                     TyrGluValLeuAlaSerThrGluSerGlnTyrAspTyrIleAsnAsn                              305310315320                                                                  MetValThrPheIleAspAsnHisAspMetAspArgPheGlnValAla                              325330335                                                                     GlySerGlyThrArgAlaThrGluGlnAlaLeuAlaLeuThrLeuThr                              340345350                                                                     SerArgGlyValProAlaIleTyrTyrGlyThrGluGlnTyrMetThr                              355360365                                                                     GlyAspGlyAspProAsnAsnArgAlaMetMetThrSerPheAsnThr                              370375380                                                                     GlyThrThrAlaTyrLysValIleGlnAlaLeuAlaProLeuArgLys                              385390395400                                                                  SerAsnProAlaIleAlaTyrGlyThrThrThrGluArgTrpValAsn                              405410415                                                                     AsnAspValLeuIleIleGluArgLysPheGlySerSerAlaAlaLeu                              420425430                                                                     ValAlaIleAsnArgAsnSerSerAlaAlaTyrProIleSerGlyLeu                              435440445                                                                     LeuSerSerLeuProAlaGlyThrTyrSerAspValLeuAsnGlyLeu                              450455460                                                                     LeuAsnGlyAsnSerIleThrValGlySerGlyGlyAlaValThrAsn                              465470475480                                                                  PheThrLeuAlaAlaGlyGlyThrAlaValTrpGlnTyrThrAlaPro                              485490495                                                                     GluThrSerProAlaIleGlyAsnValGlyProThrMetGlyGlnPro                              500505510                                                                     GlyAsnIleValThrIleAspGlyArgGlyPheGlyGlyThrAlaGly                              515520525                                                                     ThrValTyrPheGlyThrThrAlaValThrGlySerGlyIleValSer                              530535540                                                                     TrpGluAspThrGlnIleLysAlaValIleProLysValAlaAlaGly                              545550555560                                                                  LysThrGlyValSerValLysThrSerSerGlyThrAlaSerAsnThr                              565570575                                                                     PheLysSerPheAsnValLeuThrGlyAspGlnValThrValArgPhe                              580585590                                                                     LeuValAsnGlnAlaAsnThrAsnTyrGlyThrAsnValTyrLeuVal                              595600605                                                                     GlyAsnAlaAlaGluLeuGlyThrTrpAspProAsnLysAlaIleGly                              610615620                                                                     ProMetTyrAsnGlnValIleAlaLysTyrProSerTrpTyrTyrAsp                              625630635640                                                                  ValSerValProAlaGlyThrLysLeuAspPheLysPheIleLysLys                              645650655                                                                     GlyGlyGlyThrValThrTrpGluGlyGlyGlyAsnHisThrTyrThr                              660665670                                                                     ThrProAlaSerGlyValGlyThrValThrValAspTrpGlnAsn                                 675680685                                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AsnThrAlaTyrGlyTyrAlaTyrHisGlyTyrTrpMetLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      SerGlyValAsnAsnThrAlaTyrHisGlyTyrTrpProArg                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GluAsnPheGlyThrAlaAspAsp                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AlaAlaPheGlySerPheThrAsp                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AspMetLeuLeuMetValAspIleValThrAsnHisTyrGlySer                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AsnIleLysValValMetAspPheAlaProAsnHisThrAsnPro                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TATCATGGTTACTGGATGAAG21                                                       (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TATCACGGTTACTGGGCGAGG21                                                       __________________________________________________________________________

What we claim is:
 1. A method for producing a malto-oligosaccharide of apolymerization degree of 7 or more, comprising subjecting a substratemalto-oligosaccharide to transglycosylation with a variant α-amylasefrom Saccharomycopsis Fibuligera in which the tyrosine residue atposition 83 of the amino acid sequence thereof is substituted with aphenylalanine, a tryptophan, a leucine, or an asparagine residue.